Human antibodies that bind cd22 and uses thereof

ABSTRACT

The present disclosure provides isolated monoclonal antibodies that specifically bind to CD22 with high affinity, particularly human monoclonal antibodies. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Antibody-partner molecule conjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of this disclosure are also provided. This disclosure also provides methods for detecting CD22, as well as methods for treating various cancers and inflammatory and autoimmune disorders using an anti-CD22 antibody of this disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/772,063, filed Feb. 20, 2013, which is Divisional of U.S. applicationSer. No. 12/517,183, filed Feb. 17, 2010 (now U.S. Pat. No. 8,481,683,issued Jul. 9, 2013), which is a national stage of InternationalApplication Serial No. PCT/US2007/086152, filed Nov. 30, 2007, whichclaims priority of U.S. Provisional Application Ser. No. 60/868,231,filed on Dec. 1, 2006, each of which is herein incorporated byreference.

BACKGROUND

CD22 is a cell-surface type I glycoprotein of the sialoadhesin family.CD22 is also known in the art as BL-CAM, B3, Leu-14 and Lyb-8, amongother names. CD22 was initially characterized by the antibodiesanti-S-HCL-1 (Schwarting, R. et al. (1985) Blood 65:974-983), HD39(Dorken, B. et al. (1986) J. Immunol. 136:4470-4479) and RFB4 (Campana,D. et al. (1985) J. Immunol. 134:1524-1530). CD22 has been establishedas a lectin-like adhesion molecule that binds alpha2,6-linked sialicacid-bearing ligands and as a regulator of B cell antigen receptor (BCR)signaling. Structurally, there are several splice variants of CD22 thatexist, but the predominant form in humans has an extracellular regioncontaining seven immunoglobulin-like domains.

CD22 has been shown to be specifically expressed by B lymphocytes and isfunctionally important as a negative regulator of B lymphocyteactivation (reviewed by Nitschke, L. (2005) Curr. Opin. Immunol.17:290-297 and Tedder, T. F. et al (2005) Adv. Immunol. 88:1-50). Instudies that utilized gene-targeted mice that expressed mutant CD22molecules that do not interact with alpha2,6-linked sialic acid ligands,it was determined that certain functions (such as expression of cellsurface CD22, IgM and MHC Class II on mature B cells, maintenance ofmarginal zone B cell populations, optimal B cell antigenreceptor-induced proliferation and B cell turnover rates) were regulatedby CD22 ligand binding, whereas other functions (such as CD22phosphorylation, CD22 negative regulation of calcium mobilization afterBCR ligation, recruitment of SHP-1 to CD22 and B cell migration) did notrequire ligand engagement (Poe, J. C. et al. (2004) Nat. Immunol.5:1078-1087).

CD22 is considered to be an inhibitory co-receptor that downmodulatesBCR signalling by setting a signalling threshold that preventsoverstimulation of B cells. Activation of such an inhibitory co-receptoroccurs by phosphorylation on cytoplasmic ITIMs (immunoreceptortyrosine-based inhibition motifs), followed by recruitment of thetyrosine phosphatase SHP-1 or the lipid phosphatase SHIIP (reviewed inby Nitschke, L. (2005) Curr. Opin. Immunol. 17:290-297). Additionally,CD22 has been found to play a central role in a regulatory loopcontrolling the CD19/CD21-Src-family protein tyrosine kinase (PTK)amplification pathway that regulates basal signaling thresholds andintensifies Src-family kinase activation after BCR ligation (reviewed inTedder, T. F. et al (2005) Adv. Immunol. 88:1-50).

Approximately 60-80% of B cell malignancies express CD22, thereby makingit a potential target for passive immunotherapy (see e.g., Cesano, A.and Gayko, U. (2003) Semin. Oncol. 30:253-257). Moreover, selectivemodulation of B cell activity via targeting of CD22 has been suggestedas a means for treatment of autoimmune diseases (see e.g., Steinfeld, S.D. and Youinou, P. (2006) Expert. Opin. Biol. Ther. 6:943-949). Ahumanized anti-CD22 monoclonal antibody, epratuzumab, has been described(Coleman, M. et al. (2003) Clin. Cancer Res. 9:3991S-3994S). However,additional anti-CD22 reagents are still needed.

SUMMARY

The present disclosure provides isolated monoclonal antibodies, inparticular human monoclonal antibodies, that bind to human CD22 and thatexhibit numerous desirable properties. These properties include highaffinity binding to CD22, the ability to internalize into CD22+ cells,the ability to mediate antibody dependent cellular cytotoxicity (ADCC),the ability to enhance cell death of Ramos cells induced by B cellreceptor (BCR) stimulation, and/or inhibits growth of CD22-expressingcells in vivo when conjugated to a cytotoxin. The antibodies of theinvention can be used, for example, to treat CD22+ B cell malignanciesand/or to treat various inflammatory or autoimmune disorders.

In one aspect, the instant disclosure pertains to an isolated humanmonoclonal antibody, or an antigen-binding portion thereof, wherein theantibody binds to human CD22 and exhibits at least one of the followingproperties:

(a) internalizes into CD22⁺ cells;

(b) exhibits antibody dependent cellular cytotoxicity (ADCC) againstCD22⁺ cells;

(c) enhances cell death of Ramos cells induced by B cell receptor (BCR)stimulation; and

(d) inhibits growth of CD22-expressing cells in vivo when conjugated toa cytotoxin.

In another embodiment, the antibody exhibits at least two of properties(a), (b), (c) and (d). In yet another embodiment, the antibody exhibitsthree of properties (a), (b), (c) and (d). In another embodiment, theantibody exhibits all four of properties (a), (b), (c), and (d). Incertain embodiments, the antibody does not have a directanti-proliferative effect on Ramos cells. In certain embodiments, theantibody does not induce calcium flux in Ramos cells. In certainembodiments, the antibody does not mediate complement dependentcytotoxicity (CDC) on Ramos cells. Preferably, the antibody binds tohuman CD22 with high affinity, e.g., with a K_(D) of 1×10⁻⁷ M or less ora K_(D) of 1×10⁻⁸ M or less or a K_(D) of 1×10⁻⁹ M or less or a K_(D) of1×10⁻¹⁰ or less or a K_(D) of 7×10⁻¹¹ or less.

In another aspect, the invention pertains to an isolated humanmonoclonal antibody, or antigen binding portion thereof, wherein theantibody cross-competes for binding to CD22 with a reference antibody,wherein the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:31 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:35; or

(b) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:32 or 61 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:36; or

(c) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:33 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:37 or 38; or

(d) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:34 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:39 or 40; or

(e) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:81 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:84 or 85; or

(f) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:82 or 83 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:86.

wherein the antibody specifically binds human CD22

In yet another aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 7-4.1 gene, a human V_(H) 4-34 gene, a human V_(H) 5-51 gene, or ahuman VH 1-69 gene, wherein the antibody specifically binds human CD22.In yet another aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a lightchain variable region that is the product of or derived from a humanV_(λ) 2b2 gene, a human V_(K) L6 gene, a human V_(K) A27 gene, a humanV_(K) A10 gene, or a human L18 gene, wherein the antibody specificallybinds human CD22. In still another aspect, the invention pertains to anisolated antibody, or antigen-binding portion thereof, comprising:

(a) a heavy chain variable region that is the product of or derived froma human V_(H) 7-4.1 gene and a light chain variable region that is theproduct of or derived from a human V_(λ) 2b2 gene; or

(b) a heavy chain variable region that is the product of or derived froma human V_(H) 4-34 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene; or

(c) a heavy chain variable region that is the product of or derived froma human V_(H) 5-51 gene and a light chain variable region that is theproduct of or derived from a human V_(K) A27 or A10 gene;

(d) a heavy chain variable region that is the product of or derived froma human V_(H) 1-69 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene; or

(e) a heavy chain variable region that is the product of or derived froma human V_(H) 1-69 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L18 or A27 gene;

wherein the antibody specifically binds human CD22.

In another aspect, this disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising:

-   -   a heavy chain variable region that comprises CDR1, CDR2, and        CDR3 sequences;    -   and a light chain variable region that comprises CDR1, CDR2, and        CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 9-12 and 69-71, and conservative modifications thereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 25-30, 78-80, and conservative modifications thereof; and

(c) the antibody binds to human CD22.

In preferred embodiments, this antibody also has one or more of thefollowing characteristics: internalizes into CD22+ cells, mediates ADCCactivity and/or enhances cell death of Ramos cells induced by BCRstimulation, and/or inhibits growth of CD22-expressing cells in vivowhen conjugated to a cytotoxin.

Preferably, the heavy chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 5-8, 60, 66-68, and conservative modificationsthereof; and the light chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 19-24, 75-77, and conservative modificationsthereof.

Preferably, the heavy chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 1-4, 63-65, and conservative modificationsthereof; and the light chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 13-18, 72-74, and conservative modificationsthereof.

A preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:5;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:9;

(d) a light chain variable region CDR1 comprising SEQ ID NO:13;

(e) a light chain variable region CDR2 comprising SEQ ID NO:19; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:25.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:2;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:6 or 60;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 14;

(e) a light chain variable region CDR2 comprising SEQ ID NO:20; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:26.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:3;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:7;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 11;

(d) a light chain variable region CDR1 comprising SEQ ID NO:15 or 16;

(e) a light chain variable region CDR2 comprising SEQ ID NO:21 or 22;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:27 or 28.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:4;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:8;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

(d) a light chain variable region CDR1 comprising SEQ ID NO:17 or 18;

(e) a light chain variable region CDR2 comprising SEQ ID NO:23 or 24;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:29 or 30.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:63;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:66;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:69;

(d) a light chain variable region CDR1 comprising SEQ ID NO:72 or 73;

(e) a light chain variable region CDR2 comprising SEQ ID NO:75 or 76;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:78 or 79.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:64 or 65;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:67 or 68;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:70 or 71;

(d) a light chain variable region CDR1 comprising SEQ ID NO:74;

(e) a light chain variable region CDR2 comprising SEQ ID NO:77; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:80.

Other preferred antibodies of this disclosure, or antigen bindingportions thereof, comprise:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        31-34, 61 and 81-83; and    -   (b) a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 35-40        and 84-86;        -   wherein the antibody specifically binds human CD22.

A preferred combination comprises: (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:31; and (b) a lightchain variable region comprising the amino acid sequence of SEQ IDNO:35.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NOS: 32 or 61; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO:36.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:33; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO:37 or 38.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:34; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO:39 or 40.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:81; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO:84 or 85.

Another preferred combination comprises: (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:82 or 83; and (b)a light chain variable region comprising the amino acid sequence of SEQID NO:86.

In another aspect of this disclosure, antibodies, or antigen-bindingportions thereof, are provided that compete for binding to CD22 with anyof the aforementioned antibodies.

The antibodies of this disclosure can be, for example, full-lengthantibodies, for example of an IgG1 or IgG4 isotype. Alternatively, theantibodies can be antibody fragments, such as Fab, Fab′ or Fab′2fragments, or single chain antibodies.

This disclosure also provides an immunoconjugate comprising an antibodyof this disclosure, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. In aparticularly preferred embodiment, the invention provides animmunoconjugate comprising an antibody of this disclosure, orantigen-binding portion thereof, linked to the compound “Cytotoxin A”(e.g., via a thiol linkage). This disclosure also provides a bispecificmolecule comprising an antibody, or antigen-binding portion thereof, ofthis disclosure, linked to a second functional moiety having a differentbinding specificity than said antibody, or antigen binding portionthereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of this disclosure and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of this disclosure are also encompassed by thisdisclosure, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Methods for preparinganti-CD22 antibodies using the host cells comprising such expressionvectors are also provided and may include the steps of (i) expressingthe antibody in the host cell and (ii) isolating the antibody from thehost cell.

Another aspect of this disclosure pertains to methods of inhibitinggrowth of a CD22-expressing tumor cell. The method comprises contactingthe CD22-expressing tumor cell with an antibody, or antigen-bindingportion thereof, of the invention such that growth of theCD22-expressing tumor cell is inhibited. The tumor cell can be, forexample, a B cell lymphoma, such as a non-Hodgkin's lymphoma. In certainembodiments, the antibody, or antigen-binding portion thereof, isconjugated to a therapeutic agent, such as a cytotoxin.

Another aspect of this disclosure pertains to methods of treating aninflammatory or autoimmune disorder in a subject. The method comprisesadministering to the subject an antibody, or antigen-binding portionthereof, of the invention such that the inflammatory or autoimmunedisorder in the subject is treated. The autoimmune disorder can be, forexample, systemic lupus erythematosus or rheumatoid arthritis.

The present disclosure also provides isolated anti-CD22 antibody-partnermolecule conjugates that specifically bind to CD22 with high affinity,particularly those comprising human monoclonal antibodies. Certain ofsuch antibody-partner molecule conjugates are capable of beinginternalized into CD22-expressing cells and are capable of mediatingantibody dependent cellular cytotoxicity. This disclosure also providesmethods for treating cancers, such as a B cell lymphoma, such as anon-Hodgkin's lymphoma, using an anti-CD22 antibody-partner moleculeconjugate disclosed herein.

In another aspect, the invention provides a method of treating aninflammatory or autoimmune disorder in a subject. The method comprisesadministering to the subject an antibody, or antigen-binding portionthereof, of the invention such that the inflammatory or autoimmunedisorder in the subject is treated. Non-limiting examples of preferredautoimmune disorders include systemic lupus erythematosus and rheumatoidarthritis. Other examples of autoimmune disorders include inflammatorybowel disease (including ulcerative colitis and Crohn's disease), Type Idiabetes, multiple sclerosis, Sjogren's syndrome, autoimmune thyroiditis(including Grave's disease and Hashimoto's thyroiditis), psoriasis andglomerulonephritis.

Compositions comprising an antibody, or antigen-binding portion thereof,conjugated to a partner molecule of this disclosure are also provided.Partner molecules that can be advantageously conjugated to an antibodyin an antibody partner molecule conjugate as disclosed herein include,but are not limited to, molecules as drugs, toxins, marker molecules(e.g., radioisotopes), proteins and therapeutic agents. Compositionscomprising antibody-partner molecule conjugates and pharmaceuticallyacceptable carriers are also disclosed herein.

In one aspect, such antibody-partner molecule conjugates are conjugatedvia chemical linkers. In some embodiments, the linker is a peptidyllinker, and is depicted herein as (L4)p-F-(L1)m. Other linkers includehydrazine and disulfide linkers, and is depicted herein as (L4)p-H-(L1)mor (L4)p-J-(L1)m, respectively. In addition to the linkers beingattached to the partner, the present invention also provides cleavablelinker arms that are appropriate for attachment to essentially anymolecular species.

In another aspect, the invention pertains to a method of inhibitinggrowth of a CD22-expressing tumor cell. The method comprises contactingthe CD22-expressing tumor cell with an antibody-partner moleculeconjugate of the disclosure such that growth of the CD22-tumor cell isinhibited. In a preferred embodiment, the partner molecule is atherapeutic agent, such as a cytotoxin. Particularly preferredCD22-expressing tumor cells are B cell lymphomas, such as non-Hodgkin'slymphoma. Other types of CD22-expressing tumor cells include Burkitt'slymphomas and B cell chronic lymphocytic leukemias. In still otherembodiments, the CD22-expressing tumor cell is from a cancer selectedfrom the group consisting of Burkitt's lymphomas and B cell chroniclymphocytic leukemias.

In another aspect, the invention pertains to a method of treating cancerin a subject. The method comprises administering to the subject anantibody-partner molecule conjugate of the disclosure such that thecancer is treated in the subject. In a preferred embodiment, the partnermolecule is a therapeutic agent, such as a cytotoxin. Particularlypreferred cancers for treatment are B cell lymphomas, such as anon-Hodgkin's lymphoma. Other types of cancers include Burkitt'slymphomas and B cell chronic lymphocytic leukemias.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, Genbankentries, patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO:41) and amino acidsequence (SEQ ID NO:31) of the heavy chain variable region of the 12C5human monoclonal antibody. The CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:5)and CDR3 (SEQ ID NO:9) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO:45) and amino acidsequence (SEQ ID NO:35) of the lambda light chain variable region of the12C5 human monoclonal antibody. The CDR1 (SEQ ID NO:13), CDR2 (SEQ IDNO:19) and CDR3 (SEQ ID NO:25) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO:42) and amino acidsequence (SEQ ID NO:32) of the heavy chain variable region of the 19A3human monoclonal antibody, and the nucleotide sequence and amino acidsequence of the heavy chain variable region of the CD22.1 recombinantantibody. The sequences of the heavy chain variable region of 19A3 areidentical to that of CD22.1. The CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:6)and CDR3 (SEQ ID NO:10) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO:46) and amino acidsequence (SEQ ID NO:36) of the kappa light chain variable region of the19A3 human monoclonal antibody, and the nucleotide and amino acidsequences of the kappa light chain variable region of the CD22.1recombinant human monoclonal antibody. The sequences of the kappa lightchain variable region of both CD22.1 and CD22.2 are identical to thoseof 19A3. The CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:20) and CDR3 (SEQ IDNO:26) regions are delineated and the V and J germline derivations areindicated.

FIG. 2C shows the nucleotide sequence (SEQ ID NO:62) and amino acidsequence (SEQ ID NO:61) of the heavy chain variable region variableregion of the CD22.2 recombinant human monoclonal antibody. The CDR1(SEQ ID NO:2), CDR2 (SEQ ID NO:60) and CDR3 (SEQ ID NO:10) regions aredelineated and the V and J germline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO:43) and amino acidsequence (SEQ ID NO:33) of the heavy chain variable region of the 16F7human monoclonal antibody. The CDR1 (SEQ ID NO:3), CDR2 (SEQ ID NO:7)and CDR3 (SEQ ID NO: 11) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO:47) and amino acidsequence (SEQ ID NO:37) of the V_(K).1 kappa light chain variable regionof the 16F7 human monoclonal antibody. The CDR1 (SEQ ID NO:15), CDR2(SEQ ID NO 21:) and CDR3 (SEQ ID NO 27:) regions are delineated and theV and J germline derivations are indicated.

FIG. 3C shows the nucleotide sequence (SEQ ID NO:48) and amino acidsequence (SEQ ID NO:38) of the V_(K).2 kappa light chain variable regionof the 16F7 human monoclonal antibody. The CDR1 (SEQ ID NO:16), CDR2(SEQ ID NO:22) and CDR3 (SEQ ID NO:28) regions are delineated and the Vand J germline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO:44) and amino acidsequence (SEQ ID NO:34) of the heavy chain variable region of the 23C6human monoclonal antibody. The CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:8)and CDR3 (SEQ ID NO:12) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO:49) and amino acidsequence (SEQ ID NO:39) of the V_(K).1 kappa light chain variable regionof the 23C6 human monoclonal antibody. The CDR1 (SEQ ID NO:17), CDR2(SEQ ID NO:23) and CDR3 (SEQ ID NO:29) regions are delineated and the Vand J germline derivations are indicated.

FIG. 4C shows the nucleotide sequence (SEQ ID NO:50) and amino acidsequence (SEQ ID NO:40) of the V_(K).2 kappa light chain variable regionof the 23C6 human monoclonal antibody. The CDR1 (SEQ ID NO:18), CDR2(SEQ ID NO:24) and CDR3 (SEQ ID NO:30) regions are delineated and the Vand J germline derivations are indicated.

FIG. 5A shows the alignment of the amino acid sequence of the heavychain variable regions of 12C5 (SEQ ID NO:31) with the human germlineV_(H) 7-4.1 amino acid sequence (SEQ ID NO:51).

FIG. 5B shows the alignment of the amino acid sequence of the lightchain variable region of 12C5 (SEQ ID NO:35) with the human germlineV_(λ) 2b2 amino acid sequence (SEQ ID NO:55).

FIG. 6A shows the alignment of the amino acid sequence of the heavychain variable regions of 19A3/CD22.1 (SEQ ID NO:32) with the humangermline V_(H) 4-34 amino acid sequence (SEQ ID NO:52).

FIG. 6B shows the alignment of the amino acid sequence of the lightchain variable regions of 19A3/CD22.1/CD22.2 (SEQ ID NO:36) with thehuman germline V_(K) L6 amino acid sequence (SEQ ID NO:56).

FIG. 6C shows the alignment of the amino acid sequence of the heavychain variable region of CD22.2 (SEQ ID NO:61) with the human germlineV_(H) 4-34 amino acid sequence (SEQ ID NO:52).

FIG. 7A shows the alignment of the amino acid sequence of the heavychain variable regions of 16F7 (SEQ ID NO:33) with the human germlineV_(H) 5-51 amino acid sequence (SEQ ID NO:53).

FIG. 7B shows the alignment of the amino acid sequence of the V_(K).1light chain variable region of 16F7 (SEQ ID NO:37) with the humangermline V_(K) A27 amino acid sequence (SEQ ID NO:57).

FIG. 7C shows the alignment of the amino acid sequence of the V_(K).2light chain variable region of 16F7 (SEQ ID NO:38) with the humangermline V_(K) A10 amino acid sequence (SEQ ID NO:57).

FIG. 8A shows the alignment of the amino acid sequence of the heavychain variable regions of 23C6 (SEQ ID NO:34) with the human germlineV_(H) 1-69 amino acid sequence (SEQ ID NO:54).

FIG. 8B shows the alignment of the amino acid sequence of the V_(K).1light chain variable region of 23C6 (SEQ ID NO:39) and the V_(K).2 lightchain variable region of 23C6 (SEQ ID NO:40) with the human germlineV_(K) L6 amino acid sequence (SEQ ID NO:56).

FIG. 9 is a bar graph showing the internalization of anti-CD22 humanantibodies 12C5, 19A3, 16F7 and 23C6 into Raji cells.

FIG. 10A is a graph showing ADCC activity (as measured by % lysis) ofanti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6 against Daudicells.

FIG. 10B is a graph showing ADCC activity (as measured by % lysis) ofanti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6 against Raji cells.

FIG. 11 is a bar graph showing the effect of immobilized anti-CD22 humanantibodies 12C5, 19A3, 16F7 and 23C6 on BCR-stimulated Ramos cells, asmeasured by % cell death.

FIG. 12 is a graph showing CD22 ECD binding by anti-CD22 recombinanthuman antibodies CD22.1 and CD22.2 as compared to that of the 19A3parent human antibody.

FIG. 13 is a graph showing binding of CD22 expressed on the surface ofCHO cells by the by anti-CD22 recombinant human antibodies CD22.1 andCD22.2.

FIG. 14 is a graph showing binding of CD22 expressed on the surface ofRaji cells by the by anti-CD22 recombinant human antibodies CD22.1 andCD22.2.

FIG. 15 is a bar graph showing binding of the CD22 ECD amino-terminaldomains 1 and 2 by anti-CD22 antibodies 12C5, 19A3, 16F7 and 23C6, andby recombinant human antibodies CD22.1 and CD22.2.

FIG. 16 shows the in-vivo effect of antibody-drug conjugatesCD22.1-Cytotoxin A and CD22.2-Cytotoxin A on Raji-cell tumor size inSCID mice.

FIG. 17A shows the nucleotide sequence (SEQ ID NO:87) and amino acidsequence (SEQ ID NO:81) of the 4G6 human antibody. The CDR1 (SEQ IDNO:63), CDR2 (SEQ ID NO:66) and CDR3 (SEQ ID NO:69) regions aredelineated and the V, D and J germline derivations are indicated.

FIG. 17B shows the nucleotide sequence (SEQ ID NO:90) and amino acidsequence (SEQ ID NO:84) of the V_(K)1 kappa light chain variable regionof the 4G6 human monoclonal antibody. The CDR1 (SEQ ID NO:72), CDR2 (SEQID NO:75) and CDR3 (SEQ ID NO:78) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 17C shows the nucleotide sequence (SEQ ID NO:91) and amino acidsequence (SEQ ID NO:85) of the V_(K)2 kappa light chain variable regionof the 4G6 human monoclonal antibody. The CDR1 (SEQ ID NO:73), CDR2 (SEQID NO:76) and CDR3 (SEQ ID NO:79) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 18A shows the nucleotide sequence (SEQ ID NO:88) and amino acidsequence (SEQ ID NO:82) of the V_(H)1 heavy chain variable region of the21F6 human monoclonal antibody. The CDR1 (SEQ ID NO:64), CDR2 (SEQ IDNO:67) and CDR3 (SEQ ID NO:70) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 18B shows the nucleotide sequence (SEQ ID NO:89) and amino acidsequence (SEQ ID NO:83) of the V_(H)2 heavy chain variable region of the21F6 human monoclonal antibody. The CDR1 (SEQ ID NO:65), CDR2 (SEQ IDNO:68) and CDR3 (SEQ ID NO:71) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 18C shows the nucleotide sequence (SEQ ID NOs:92) and amino acidsequence (SEQ ID NO:86) of the kappa light chain variable region of the21F6 human monoclonal antibody. The CDR1 (SEQ ID NO:74), CDR2 (SEQ IDNO:77) and CDR3 (SEQ ID NO:80) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 19A shows the alignment of the amino acid sequence of the heavychain variable regions of 4G6 (SEQ ID NO:81) with the human germlineV_(H) 1-69 amino acid sequence (SEQ ID NO:54).

FIG. 19B shows the alignment of the amino acid sequence of the V_(K)1kappa light chain variable region of 4G6 (SEQ ID NO:84) with the humangermline V_(K) L18 amino acid sequence (SEQ ID NO:93).

FIG. 19C shows the alignment of the amino acid sequence of the V_(K)2kappa light chain variable region of 4G6 (SEQ ID NO:85) with the humangermline V_(K) A27 amino acid sequence (SEQ ID NO:57).

FIG. 20A shows the alignment of the amino acid sequence of the V_(H)1heavy chain variable regions of 21F6 (SEQ ID NO:82) with the humangermline V_(H) 4-34 amino acid sequence (SEQ ID NO:52).

FIG. 20B shows the alignment of the amino acid sequence of the V_(H)2heavy chain variable regions of 21F6 (SEQ ID NO:83) with the humangermline V_(H) 4-34 amino acid sequence (SEQ ID NO:52).

FIG. 20C shows the alignment of the amino acid sequence of the kappalight chain variable region of 21F6 (SEQ ID NO:86) with the humangermline V_(K) L6 amino acid sequence (SEQ ID NO:56).

FIG. 21 is a graph showing binding of CD22 expressed on the surface ofCHO cells by the anti-CD22 human antibody 4G6.

FIG. 22 is a graph showing binding of CD22 expressed on the surface ofCHO cells by anti-CD22 human antibody 21F6.

FIG. 23 is a graph showing binding of CD22 expressed on the surface ofRaji cells by anti-CD22 human antibody 21F6.

DETAILED DESCRIPTION OF THIS DISCLOSURES

The present disclosure relates to isolated monoclonal antibodies,particularly human monoclonal antibodies that bind specifically to humanCD22 with high affinity. In certain embodiments, the antibodies of thisdisclosure are derived from particular heavy and light chain germlinesequences and/or comprise particular structural features such as CDRregions comprising particular amino acid sequences. This disclosureprovides isolated antibodies, immuno-partner molecule conjugates,bispecific molecules, affibodies, domain antibodies, nanobodies andunibodies, methods of making said molecules, and pharmaceuticalcompositions comprising said molecules and pharmaceutical carriers. Theinvention also relates to methods of using the molecules, such as todetect CD22, as well as to modulate B cell activity in diseases ordisorders associated with expression of CD22 or involving B cellregulation, such as CD22+ tumors and inflammatory or autoimmunedisorders. This disclosure also provides methods of using the anti-CD22antibodies of this invention to inhibit the growth of CD22+ tumor cells,for example, to treat B cell lymphomas. Additionally, this disclosureprovides methods of using the anti-CD22 antibodies of this disclosure totreat inflammatory or autoimmune disorders.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “CD22,” “BL-CAM,” “B3,” “Leu-14” and “Lyb-8” are usedinterchangeably, and include variants, isoforms, and species homologs ofCD22. Accordingly, human antibodies of this disclosure may, in certaincases, cross-react with CD22 from species other than human. In certainembodiments, the antibodies may be completely specific for human CD22and may not exhibit species or other types of non-humancross-reactivity. The complete amino acid sequence of an exemplary humanCD22 has Genbank accession number NP_001762 (SEQ ID NO:59).

The human CD22 sequence may differ from human CD22 of SEQ ID NO:59 byhaving, for example, conserved mutations or mutations in non-conservedregions and the CD22 has substantially the same biological function asthe human CD22 of SEQ ID NO:59. For example, a biological function ofhuman CD22 is having an epitope in the extracellular domain of CD22 thatis specifically bound by an antibody of the instant disclosure or abiological function of human CD22 is modulation of BCR signalling.

A particular human CD22 sequence will generally be at least 90%identical in amino acids sequence to human CD22 of SEQ ID NO:59 andcontains amino acid residues that identify the amino acid sequence asbeing human when compared to CD22 amino acid sequences of other species(e.g., murine). In certain cases, a human CD22 may be at least 95%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence toCD22 of SEQ ID NO:59. In certain embodiments, a human CD22 sequence willdisplay no more than 10 amino acid differences from the CD22 of SEQ IDNO:59. In certain embodiments, the human CD22 may display no more than5, or even no more than 4, 3, 2, or 1 amino acid difference from theCD22 of SEQ ID NO:59. Percent identity can be determined as describedherein.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present disclosure is the CD22 protein.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H)1, C_(H)2 and C_(H)3. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., CD22). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region(see, Fundamental Immunology (Paul ed., 3.sup.rd ed. 1993); (iv) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The abbreviation “V_(K)”, as used herein, refers to the variable domainof a kappa light chain, whereas the abbreviation “V_(λ)”, as usedherein, refers to the variable domain of a lambda light chain. Theabbreviation “V_(L)”, as used herein, refers to the variable domain ofan immunoglobulin light chain and thus encompasses both V_(K) and V_(λ□)light chains.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds CD22 is substantially free of antibodies that specifically bindantigens other than CD22). An isolated antibody that specifically bindsCD22 may, however, have cross-reactivity to other antigens, such as CD22molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of this disclosure may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity, which have variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic nonhuman animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The term “antibody mimetic” is intended to refer to molecules capable ofmimicking an antibody's ability to bind an antigen, but which are notlimited to native antibody structures. Examples of such antibodymimetics include, but are not limited to, Affibodies, DARPins,Anticalins, Avimers, and Versabodies, all of which employ bindingstructures that, while they mimic traditional antibody binding, aregenerated from and function via distinct mechanisms.

As used herein, the term “partner molecule” refers to the entity whichis conjugated to an antibody in an antibody-partner molecule conjugate.Examples of partner molecules include drugs, toxins, marker molecules(including, but not limited to peptide and small molecule markers suchas fluorochrome markers, as well as single atom markers such asradioisotopes), proteins and therapeutic agents.

As used herein, an antibody that “specifically binds to human CD22” isintended to refer to an antibody that binds to human CD22 (and possiblyCD22 from one or more non-human species) but does not substantially bindto non-CD22 proteins. In certain embodiments, an antibody of the instantdisclosure specifically binds to human CD22 of SEQ ID NO:59 or a variantthereof. Preferably, the antibody binds to human CD22 with a K_(D) of1×10⁻⁷ M or less, more preferably 1×10⁻⁸ M or less, more preferably5×10⁻⁹ M or less, more preferably 1×10⁻⁹ M or less, even more preferably5×10⁻¹⁰ M or less, and even more preferably 7×10⁻¹¹ or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ Mor less, even more preferably 1×10⁻⁸ M or less, even more preferably5×10⁻⁹ M or less and even more preferably 1×10⁻⁹ M or less for a targetantigen. However, “high affinity” binding can vary for other antibodyisotypes. For example, “high affinity” binding for an IgM isotype refersto an antibody having a K_(D) of 10⁻⁶ M or less, more preferably 10⁻⁷ Mor less, even more preferably 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

The symbol “—”, whether utilized as a bond or displayed perpendicular toa bond, indicates the point at which the displayed moiety is attached tothe remainder of the molecule, solid support, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups, whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, and S, and wherein the nitrogen,carbon and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, S, andSi may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). The terms “heteroalkyl” and“heteroalkylene” encompass poly(ethylene glycol) and its derivatives(see, for example, Shearwater Polymers Catalog, 2001). Still further,for alkylene and heteroalkylene linking groups, no orientation of thelinking group is implied by the direction in which the formula of thelinking group is written. For example, the formula —C(O)₂R′— representsboth —C(O)₂R′— and —R′C(O)₂—.

The term “lower” in combination with the terms “alkyl” or “heteroalkyl”refers to a moiety having from 1 to 6 carbon atoms.

The terms “alkoxy,” “alkylamino,” “alkylsulfonyl,” and “alkylthio” (orthioalkoxy) are used in their conventional sense, and refer to thosealkyl groups attached to the remainder of the molecule via an oxygenatom, an amino group, an SO₂ group or a sulfur atom, respectively. Theterm “arylsulfonyl” refers to an aryl group attached to the remainder ofthe molecule via an SO₂ group, and the term “sulfhydryl” refers to an SHgroup.

In general, an “acyl substituent” is also selected from the group setforth above. As used herein, the term “acyl substituent” refers togroups attached to, and fulfilling the valence of a carbonyl carbon thatis either directly or indirectly attached to the polycyclic nucleus ofthe compounds of the present invention.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of substituted or unsubstituted “alkyl” and substituted orunsubstituted “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbonatoms of the cyclic structures are optionally oxidized.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a substituted orunsubstituted polyunsaturated, aromatic, hydrocarbon substituent whichcan be a single ring or multiple rings (preferably from 1 to 3 rings)which are fused together or linked covalently. The term “heteroaryl”refers to aryl groups (or rings) that contain from one to fourheteroatoms selected from N, O, and S, wherein the nitrogen, carbon andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. “Aryl” and “heteroaryl” alsoencompass ring systems in which one or more non-aromatic ring systemsare fused, or otherwise bound, to an aryl or heteroaryl system.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl, and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generally referred to as “alkyl substituents”and “heteroalkyl substituents,” respectively, and they can be one ormore of a variety of groups selected from, but not limited to: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5, 6, or 7-membered ring. For example, —NR′R″ ismeant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical, the arylsubstituents and heteroaryl substituents are generally referred to as“aryl substituents” and “heteroaryl substituents,” respectively and arevaried and selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroarylring may optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆) alkyl.

As used herein, the term “diphosphate” includes but is not limited to anester of phosphoric acid containing two phosphate groups. The term“triphosphate” includes but is not limited to an ester of phosphoricacid containing three phosphate groups. For example, particular drugshaving a diphosphate or a triphosphate include:

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S) and silicon (Si).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups.

Various aspects of this disclosure are described in further detail inthe following subsections.

Anti-CD22 Antibodies

The antibodies of this disclosure are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to human CD22. Preferably, an antibody ofthe invention binds to CD22 with high affinity, for example with a K_(D)of 1×10⁻⁷ M or less.

The anti-CD22 antibodies of this disclosure preferably exhibit one ormore of the following characteristics:

(a) internalizing into CD22⁺ cells;

(b) exhibiting antibody dependent cellular cytotoxicity (ADCC) againstCD22⁺ cells;

(c) enhancing cell death of Ramos cells induced by B cell receptor (BCR)stimulation, and

(d) inhibits growth of CD22-expressing cells in vivo when conjugated toa cytotoxin.

In preferred embodiments, the antibody exhibits at least two ofproperties (a), (b), (c) and (d). In yet another embodiment, theantibody exhibits three of properties (a), (b), (c) and (d). In anotherembodiment, the antibody exhibits all four of properties (a), (b), (c),and (d).

While the anti-CD22 antibodies of the invention exhibit certainfunctional properties, in certain embodiments another feature of theantibodies is that they do not exhibit other particular functionalproperties. For example, in certain embodiments, the antibody does nothave a direct anti-proliferative effect on Ramos cells. In otherembodiments, the antibody does not induce calcium flux in Ramos cells.In yet other embodiments, the antibody does not mediate complementdependent cytotoxicity (CDC) on Ramos cells.

It is noted that it has been reported that a humanized anti-CD22antibody, epratuzumab, lacked a direct anti-proliferative effect and CDCactivity against non-Hodgkin's lymphoma cell lines yet the antibody didmediate cytotoxic effects against the cell lines by other means (seeCarnahan, J. et al. (2006) Mol. Immunol. 44:1331-1341).

Preferably, an antibody of this disclosure binds to human CD22 with aK_(D) of 1×10⁻⁷ M or less, binds to human CD22 with a K_(D) of 1×10⁻⁸ Mor less, binds to human CD22 with a K_(D) of 5×10⁻⁹ M or less, binds tohuman CD22 with a K_(D) of 3×10⁻⁹ M or less, binds to human CD22 with aK_(D) of 1×10⁻⁹ M or less, or binds to human CD22 with a K_(D) of5×10⁻¹⁰ M or less, or binds to human CD22 with a K_(D) of 1×10⁻¹⁰ orbinds to human CD22 with a K_(D) of 7×10⁻¹¹ M or less.

Standard assays to evaluate the binding affinity of the antibodiestoward human CD22 are known in the art, including for example, ELISA andBIAcore analysis with recombinant CD22 (see Example 3). The Examplesalso provide detailed descriptions of suitable assays for evaluatingantibody internalization (Example 4), ADCC activity (Example 5),enhancement of cell death induced by BCR stimulation (Example 7), directanti-proliferative effects of antibodies (Example 8), induction ofcalcium flux (Example 6), and CDC activity (Example 9), andanti-proliferative effects of antibody-drug immunoconjugates on solidtumor cell proliferation in vivo (Example 10).

Monoclonal Antibodies 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and21F6

Preferred antibodies of this disclosure are the human monoclonalantibodies 12C5, 19A3, 16F7, 23C6, 4G6 and 21F6, and the recombinanthuman monoclonal antibodies CD22.1, CD22.2, all of which were isolatedand structurally characterized as described in Examples 1 and 2.

The V_(H) amino acid sequences of 12C5, 19A3, CD22.1, CD22.2, 16F7,23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 31, 32, 61, 33, 34, 81, 82and 83, respectively, wherein the heavy chains of 19A3 and CD22.1 areidentical and correspond to SEQ ID NO:32 and the V_(H) heavy chain of21F6 correspond to either SEQ ID NO:82 or 83

The V_(L) amino acid sequences of 12C5, 19A3, CD22.1, CD22.2, 16F7,23C6, 4G6 and 21F6 are shown in SEQ ID NOs:35, 36, 37, 38, 39, 40, 84,85 and 86, respectively, wherein the kappa light chains of 19A3, CD22.1and CD22.2 are identical and correspond to SEQ ID NO:36, the kappa lightchain of 16F7 corresponds to either SEQ ID NO:37 or 38, the kappa lightchain of 23C6 corresponds to either SEQ ID NO:39 or 40, and the kappalight chain of 4G6 corresponds to either SEQ ID NO: 84 or 85.

Given that each of these antibodies can bind to CD22, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-CD22binding molecules of this disclosure. CD22 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISA or flow cytometry). Preferably,when V_(H) and V_(L) chains are mixed and matched, a V_(H) sequence froma particular V_(H)/V_(L) pairing is replaced with a structurally similarV_(H) sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:31-34,        61 and 81-83; and    -   (b) a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:35-40        and 84-86;        wherein the antibody specifically binds CD22, preferably human        CD22.

Preferred heavy and light chain combinations include:

-   -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO:35; or    -   (b) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:32 or 61, and a light chain variable        region comprising the amino acid sequence of SEQ ID NO:36; or    -   (c) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:33 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO:37 or 38; or    -   (d) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:34 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO:39 or 40; or    -   (e) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:81 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO:84 or 85; or    -   (f) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:82 or 83 or and a light chain variable        region comprising the amino acid sequence of SEQ ID NO:86.

In another aspect, this disclosure provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 12C5, 19A3,CD22.1, CD22.2, 16F7, 23C6, 4G6, 21F6, or combinations thereof.

The amino acid sequences of the V_(H) CDR1s of 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 1-4 and 63-65,respectively (wherein the CDR1s of the V_(H) sequences of 19A3, CD22.1and CD22.2 are identical and are shown in SEQ ID NO:2, and the CDR1s ofthe V_(H)1 and V_(H)2 sequences of 21F6 are identical and are shown inSEQ ID NOs: 64 and 65, respectively.

The amino acid sequences of the V_(H) CDR2s of 12C5, 19A3, CD22.1, 16F7,23C6, CD22.2, 4G6 and 21F6 are shown in SEQ ID NOs: 5-8, 60, and 66-68,respectively (wherein the CDR2s of the V_(H) sequences of 19A3 andCD22.1 are identical and are shown in SEQ ID NO:6, the CDR2 of the V_(H)sequence of CD22.2 is shown in SEQ ID NO:60, and the CDR2s of the V_(H)1and V_(H)2 sequences of 21F6 are shown in SEQ ID NOs:67 and 68).

The amino acid sequences of the V_(H) CDR3s of 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 9-12 and69-71, respectively (wherein the CDR3s of the V_(H). sequences of 19A3,CD22.1 and CD22.2 are identical and are shown in SEQ ID NO:10, and theCDR3s of the V_(H)1 and V_(H)2 sequences of 21F6 are shown in SEQ IDNOs:70 and 71).

The amino acid sequences of the V_(L) CDR1s of 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 13-18 and72-74, respectively (wherein the CDR1s of the V_(K) sequences of 19A3,CD22.1 and CD22.2 are identical, and are shown in SEQ ID NO:14, theCDR1s of the V_(K).1 and V_(K).2 sequences of 16F7 are shown in SEQ IDNOs: 15 and 16, the CDR1s of the V_(K).1 and V_(K).2 sequences of 23C6are shown in SEQ ID NOs: 17 and 18, and the CDR1s of the V_(K).1 andV_(K).2 sequences of 4G6 are shown in SEQ ID NOs 72 and 73).

The amino acid sequences of the V_(L) CDR2s of 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 19-24 and75-77, respectively (wherein the CDR2s of the V_(K) sequences of 19A3,CD22.1 and CD22.2 are identical, and are shown in SEQ ID NO:20, theCDR2s of the V_(K).1 and V_(K).2 sequences of 16F7 are shown in SEQ IDNOs: 21 and 22, the CDR2s of the V_(K).1 and V_(K).2 sequences of 23C6are shown in SEQ ID NOs: 23 and 24, and the CDR2s of the V_(K).1 andV_(K).2 sequences of 4G6 are shown in SEQ ID NOs: 75 and 76).

The amino acid sequences of the V_(L) CDR1s of 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 25-30 and78-80, respectively (wherein the CDR3s of the V_(K) sequences of 19A3,CD22.1 and CD22.2 are identical, and are shown in SEQ ID NO:26, theCDR3s of the V_(K).1 and V_(K).2 sequences of 16F7 are shown in SEQ IDNOs: 27 and 28, the CDR3s of the V_(K).1 and V_(K).2 sequences of 23C6are shown in SEQ ID NOs: 29 and 30, and the CDR3s of the V_(K).1 andV_(K).2 sequences of 4G6 are shown in SEQ ID NOs: 78 and 79).

The CDR regions are delineated using the Kabat system (Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242).

Given that each of these antibodies can bind to CD22 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(L) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(L) CDR1, CDR2, and CDR3)to create other anti-CD22 binding molecules of this disclosure. CD22binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore® analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(L) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(L) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies CDR1s of 12C5,19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6.

Accordingly, in another aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-4 and 63-65;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5-8, 60 and 66-68;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-12 and 69-71;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-18 and 72-74;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-24 and 75-77; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25-30 and 78-80;

wherein the antibody specifically binds CD22, preferably human CD22.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:5;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:9;

(d) a light chain variable region CDR1 comprising SEQ ID NO:13;

(e) a light chain variable region CDR2 comprising SEQ ID NO:19; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:25.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:2;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:6 or 60;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 14;

(e) a light chain variable region CDR2 comprising SEQ ID NO:20; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:26.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:3;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:7;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 11;

(d) a light chain variable region CDR1 comprising SEQ ID NO:15 or 16;

(e) a light chain variable region CDR2 comprising SEQ ID NO:21 or 22;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:27 or 28.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:4;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:8;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

(d) a light chain variable region CDR1 comprising SEQ ID NO:17 or 18;

(e) a light chain variable region CDR2 comprising SEQ ID NO:23 or 24;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:29 or 30.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:63;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO 66;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:69;

(d) a light chain variable region CDR1 comprising SEQ ID NO:72 or 73;

(e) a light chain variable region CDR2 comprising SEQ ID NO:75 or 76;and

(f) a light chain variable region CDR3 comprising SEQ ID NO:78 or 79.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:64 or 65;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:67 or 68;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:70 or 71;

(d) a light chain variable region CDR1 comprising SEQ ID NO:74;

(e) a light chain variable region CDR2 comprising SEQ ID NO:77; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:80.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka et al., British J. ofCancer 83(2):252-260 (2000) (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.296:833-849 (2000) (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader et al., Proc. Natl.Acad. Sci. U.S.A. 95:8910-8915 (1998) (describing a panel of humanizedanti-integrin α_(v)β₃ antibodies using a heavy and light chain variableCDR3 domain of a murine anti-integrin α_(v)β₃ antibody LM609 whereineach member antibody comprises a distinct sequence outside the CDR3domain and capable of binding the same epitope as the parent muringantibody with affinities as high or higher than the parent murineantibody); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994)(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci.U.S.A. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3sequences of three Fabs (SI-1, SI-40, and SI-32) against human placentalDNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacingthe existing heavy chain CDR3 and demonstrating that the CDR3 domainalone conferred binding specificity); Ditzel et al., J. Immunol.157:739-749 (1996) (describing grafting studies wherein transfer of onlythe heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chainof a monospecific IgG tetanus toxoid-binding Fab p313 antibody wassufficient to retain binding specificity of the parent Fab); Berezov etal., BIAjournal 8: Scientific Review 8 (2001) (describing peptidemimetics based on the CDR3 of an anti-HER2 monoclonal antibody; Igarashiet al., J. Biochem (Tokyo) 117:452-7 (1995) (describing a 12 amino acidsynthetic polypeptide corresponding to the CDR3 domain of ananti-phosphatidylserine antibody); Bourgeois et al., J. Virol 72:807-10(1998) (showing that a single peptide derived form the heavy chain CDR3domain of an anti-respiratory syncytial virus (RSV) antibody was capableof neutralizing the virus in vitro); Levi et al., Proc. Natl. Acad. Sci.U.S.A. 90:4374-8 (1993) (describing a peptide based on the heavy chainCDR3 domain of a murine anti-HIV antibody); Polymenis and Stoller, J.Immunol. 152:5218-5329 (1994) (describing enabling binding of an scFv bygrafting the heavy chain CDR3 region of a Z-DNA-binding antibody) and Xuand Davis, Immunity 13:37-45 (2000) (describing that diversity at theheavy chain CDR3 is sufficient to permit otherwise identical IgMmolecules to distinguish between a variety of hapten and proteinantigens). See also, U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382;6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185,describing patented antibodies defined by a single CDR domain. Each ofthese references is hereby incorporated by reference in its entirety.

Accordingly, the present disclosure provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domains from anantibody derived from a human or non-human animal, wherein themonoclonal antibody is capable of specifically binding to CD22. Withincertain aspects, the present disclosure provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domain from anon-human antibody, such as a mouse or rat antibody, wherein themonoclonal antibody is capable of specifically binding to CD22. Withinsome embodiments, such inventive antibodies comprising one or more heavyand/or light chain CDR3 domain from a non-human antibody (a) are capableof competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present disclosure provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a human antibody, such as, for example, a human antibody obtainedfrom a non-human animal, wherein the human antibody is capable ofspecifically binding to CD22. Within other aspects, the presentdisclosure provides monoclonal antibodies comprising one or more heavyand/or light chain CDR3 domain from a first human antibody, such as, forexample, a human antibody obtained from a non-human animal, wherein thefirst human antibody is capable of specifically binding to CD22 andwherein the CDR3 domain from the first human antibody replaces a CDR3domain in a human antibody that is lacking binding specificity for CD22to generate a second human antibody that is capable of specificallybinding to CD22. Within some embodiments, such inventive antibodiescomprising one or more heavy and/or light chain CDR3 domain from thefirst human antibody (a) are capable of competing for binding with; (b)retain the functional characteristics; (c) bind to the same epitope;and/or (d) have a similar binding affinity as the corresponding parentalfirst human antibody.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, this disclosure provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 7-4.1 gene, a human V_(H) 4-34 gene, a humanV_(H) 5-51 gene, or a human V_(H) 1-69 gene, wherein the antibodyspecifically binds CD22.

In another preferred embodiment, this disclosure provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(λ) 2b2 gene, a human V_(K) L6 gene, a human V_(K) A27 gene, ahuman V_(K) A10 gene, or a human V_(K) L18 gene, wherein the antibodyspecifically binds CD22.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable region that is theproduct of or derived from a human V_(H) 7-4.1 gene and comprises alight chain variable region that is the product of or derived from ahuman V_(λ) 2b2 gene, wherein the antibody specifically binds to CD22,preferably human CD22.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable region that is theproduct of or derived from a human V_(H) 4-34 gene and comprises a lightchain variable region that is the product of or derived from a humanV_(K) L6 gene, wherein the antibody specifically binds to CD22,preferably human CD22.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable region that is theproduct of or derived from a human V_(H) 5-51 gene and comprises a lightchain variable region that is the product of or derived from a humanV_(K) A27 or A10 gene, wherein the antibody specifically binds to CD22,preferably human CD22.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable region that is theproduct of or derived from a human V_(H) 1-69 gene and comprises a lightchain variable region that is the product of or derived from a humanV_(K) L6 gene, wherein the antibody specifically binds to CD22,preferably human CD22.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable region that is theproduct of or derived from a human V_(H) 1-69 gene and comprises a lightchain variable region that is the product of or derived from a humanV_(K) A27 or L18 gene, wherein the antibody specifically binds to CD22,preferably human CD22.

Such antibodies also may possess one or more of the functionalcharacteristics described in detail above, such as internalization intoCD22+ cells, ADCC activity against CD22+ cells and/or enhancement ofcell death of Ramos cells induced by BCR stimulation cytotoxin.

An example of an antibody having V_(H) and V_(L) of V_(H) 7-4.1 andV_(λ) 2b2, respectively, is the 12C5 antibody. An example of an antibodyhaving V_(H) and V_(L) of V_(H) 4-34 and V_(K) L6, respectively, is the19A3 antibody. Another example of an antibody having V_(H) and V_(L) ofV_(H) 4-34 and V_(K) L6, respectively, is the CD22.1 antibody. Anotherexample of an antibody having V_(H) and V_(L) of V_(H) 4-34 and V_(K)L6, respectively, wherein the V_(H) chain includes an N57Q mutation, isthe CD22.2 antibody. Another example of an antibody having V_(H) andV_(L) of V_(H) 4-34 and V_(K) L6 germline, respectively, is the 21F6antibody. An example of an antibody having V_(H) and V_(L) of V_(H) 5-51and V_(K) A27 or A10, respectively, is the 16F7 antibody. An example ofan antibody having V_(H) and V_(L) of V_(H) 1-69 and V_(K) L6,respectively, is the 23C6 antibody. An example of an antibody havingV_(H) and V_(L) of V_(H) 1-69 and V_(K) A27 or L18, respectively, is the4G6 antibody.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of this disclosure comprisesheavy and light chain variable regions comprising amino acid sequencesthat are homologous to the amino acid sequences of the preferredantibodies described herein, and wherein the antibodies retain thedesired functional properties of the anti-CD22 antibodies of thisdisclosure.

For example, this disclosure provides an isolated monoclonal antibody,or antigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs:31-34, 61 and 81-83;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs:35-40 and 84-86;

(c) the antibody specifically binds to human CD22.

Additionally or alternatively, the antibody may possess one or more ofthe following functional properties: (a) binds to human CD22 with aK_(D) of 1×10⁻⁷ M or less; (b) internalizes into CD22+ cells; (c)exhibits ADCC activity on CD22+ cells; (d) enhances cell death of Ramoscells induced by, for example, BCR stimulation; and/or (e) inhibitsgrowth of CD22-expressing cells in vivo when conjugated to a cytotoxin.

In various embodiments, the antibody can be, for example, a humanantibody, a humanized antibody or a chimeric antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs:41-44, 62, or 87-89, or SEQ ID NOs:45-50 or 90-92,followed by testing of the encoded altered antibody for retainedfunction (i.e., the functions set forth above) using the functionalassays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4: 11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases to, for example, to identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of thisdisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are useful. See www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6), or conservativemodifications thereof, and wherein the antibodies retain the desiredfunctional properties of the anti-CD22 antibodies of this disclosure. Itis understood in the art that certain conservative sequence modificationcan be made which do not remove antigen binding. See, for example,Brummell et al. (1993) Biochem 32:1180-8 (describing mutational analysisin the CDR3 heavy chain domain of antibodies specific for Salmonella);de Wildt et al. (1997) Prot. Eng. 10:835-41 (describing mutation studiesin anti-UA1 antibodies); Komissarov et al. (1997) J. Biol. Chem.272:26864-26870 (showing that mutations in the middle of HCDR3 led toeither abolished or diminished affinity); Hall et al. (1992) J. Immunol.149:1605-12 (describing that a single amino acid change in the CDR3region abolished binding activity); Kelley and O'Connell (1993) Biochem.32:6862-35 (describing the contribution of Tyr residues in antigenbinding); Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 (describingthe effect of hydrophobicity in binding) and Beers et al. (2000) Clin.Can. Res. 6:2835-43 (describing HCDR3 amino acid mutants).

Accordingly, this disclosure provides an isolated monoclonal antibody,or antigen binding portion thereof, comprising a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 9-12, 69-71 and conservative modifications thereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 25-30, 79-80, and conservative modifications thereof; and

(c) the antibody specifically binds to human CD22.

Additionally or alternatively, the antibody may possess one or more ofthe following functional properties: (a) binds to human CD22 with aK_(D) of 1×10⁻⁷ M or less; (b) internalizes into CD22+ cells; (c)exhibits ADCC activity on CD22+ cells; and/or (d) enhances cell death ofRamos cells induced by BCR stimulation; and/or (e) inhibits growth ofCD22-expressing cells in vivo when conjugated to a cytotoxin.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs:5-8, 60, 66-68, and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 19-24, 75-77, and conservativemodifications thereof. In another preferred embodiment, the heavy chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 1-4,63-65, and conservative modifications thereof; and the light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 13-18,72-74, and conservative modifications thereof.

In various embodiments, the antibody can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of this disclosure by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody ofthis disclosure can be replaced with other amino acid residues from thesame side chain family and the altered antibody can be tested forretained function (i.e., the functions set forth above) using thefunctional assays described herein.Antibodies that Bind to the Same Epitope as Anti-CD22 Antibodies

In another embodiment, this disclosure provides antibodies that bind tothe same epitope on human CD22 that are recognized by any of theanti-CD22 monoclonal antibodies of this disclosure (i.e., antibodiesthat have the ability to cross-compete for binding to CD22 with any ofthe monoclonal antibodies of this disclosure). In preferred embodiments,the reference antibody for cross-competition studies can be themonoclonal antibody 12C5 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs:31 and 35, respectively), or the monoclonal antibody 19A3 orthe monoclonal antibody CD22.1 or the monoclonal antibody CD22.2 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs:32/61 and 36,respectively) or the monoclonal antibody 16F7 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs:33 and 37/38, respectively) or themonoclonal antibody 23C6 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs:34 and 39/40, respectively), or the monoclonal antibody 4G6(having V_(H) and V_(L) sequences as shown in SEQ ID NOs:81 and 84/85,respectively) or the monoclonal antibody 21F6 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 82/83 and 86, respectively).

Such cross-competing antibodies can be identified based on their abilityto cross-compete with 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and21F6 in standard CD22 binding assays. For example, standard ELISA assayscan be used in which recombinant CD22 is immobilized on the plate, oneof the antibodies is fluorescently labeled and the ability ofnon-labeled antibodies to compete off the binding of the labeledantibody is evaluated. Additionally or alternatively, BIAcore analysiscan be used to assess the ability of the antibodies to cross-compete, asdescribed in Example 3 (regarding the epitope grouping of 12C5, 19A3,CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6). The ability of a testantibody to inhibit the binding of, for example, 12C5, 19A3, CD22.1,CD22.2, 16F7, 23C6, 4G6 and 21F6 to human CD22 demonstrates that thetest antibody can compete with 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6,4G6 and 21F6 for binding to human CD22 and thus binds to the sameepitope on human CD22 as is recognized by 12C5, 19A3, CD22.1, CD22.2,16F7, 23C6, 4G6 and 21F6. As described in detail in Example 3, theantibodies 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6, eachbind to a distinct epitope on CD22 and thus belong to distinct epitopegroups. In a preferred embodiment, the antibody that binds to the sameepitope on 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 is ahuman monoclonal antibody. Such human monoclonal antibodies can beprepared and isolated as described in the Examples.

Engineered and Modified Antibodies

An antibody of this disclosure further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variableregions of antibodies. Antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of this disclosure pertains to anisolated monoclonal antibody, or antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-4 and 63-65, SEQ ID NOs:5-8, 60, and 66-68,and SEQ ID NOs:9-12 and 69-71, respectively; and a light chain variableregion comprising CDR1, CDR2, and CDR3 sequences comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 13-18and 72-74, SEQ ID NOs:19-24 and 75-77, and SEQ ID NOs:25-30 and 78-80,respectively. Thus, such antibodies contain the V_(H) and V_(L) CDRsequences of monoclonal antibodies 12C5, 19A3, CD22.1, CD22.2, 16F7,23C6, 4G6 and 21F6, yet may contain different framework sequences fromthese antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference. As another example, the germline DNAsequences for human heavy and light chain variable region genes can befound in the Genbank database. For example, the following heavy chaingermline sequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank accession numbers: 1-69 (NG_0010109, NT_024637 andBC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 andNT_024637). As another example, the following heavy chain germlinesequences found in the HCo12 HuMAb mouse are available in theaccompanying Genbank accession numbers: 1-69 (NG_0010109, NT_024637 andBC070333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 andNT_024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research25:3389-3402), which is well known to those skilled in the art. BLAST isa heuristic algorithm in that a statistically significant alignmentbetween the antibody sequence and the database sequence is likely tocontain high-scoring segment pairs (HSP) of aligned words. Segment pairswhose scores cannot be improved by extension or trimming is called ahit. Briefly, the nucleotide sequences of VBASE origin(http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated and theregion between and including FR1 through FR3 framework region isretained. The database sequences have an average length of 98 residues.Duplicate sequences which are exact matches over the entire length ofthe protein are removed. A BLAST search for proteins using the programblastp with default, standard parameters except the low complexityfilter, which is turned off, and the substitution matrix of BLOSUM62,filters for top 5 hits yielding sequence matches. The nucleotidesequences are translated in all six frames and the frame with no stopcodons in the matching segment of the database sequence is consideredthe potential hit. This is in turn confirmed using the BLAST programtblastx, which translates the antibody sequence in all six frames andcompares those translations to the VBASE nucleotide sequencesdynamically translated in all six frames.

The identities are exact amino acid matches between the antibodysequence and the protein database over the entire length of thesequence. The positives (identities+substitution match) are notidentical but amino acid substitutions guided by the BLOSUM62substitution matrix. If the antibody sequence matches two of thedatabase sequences with same identity, the hit with most positives wouldbe decided to be the matching sequence hit.

Preferred framework sequences for use in the antibodies of thisdisclosure are those that are structurally similar to the frameworksequences used by selected antibodies of this disclosure, e.g., similarto the V_(H) 7-4.1 (SEQ ID NO:51), V_(H) 4-34 (SEQ ID NO:52), V_(H) 5-51(SEQ ID NO:53), or V_(H) 1-69 (SEQ ID NO:54) framework sequences and/orthe V_(λ) 2b2 (SEQ ID NO:55), V_(K) L6 (SEQ ID NO:56), V_(K) A27 (SEQ IDNO:57), V_(K) A10 (SEQ ID NO:58), or V_(K) L18 (SEQ ID NO:93) frameworksequences used by preferred monoclonal antibodies of this disclosure.The V_(H) CDR1, CDR2, and CDR3 sequences, and the V_(K) CDR1, CDR2, andCDR3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the instant disclosure providesisolated anti-CD22 monoclonal antibodies, or antigen binding portionsthereof, comprising a heavy chain variable region comprising: (a) aV_(H) CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4 or 63-65, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 1-4 or 63-65; (b) a V_(H) CDR2region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:5-8, 60 or 66-68, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs:5-8, 60 or 66-68; (c) a V_(H)CDR3 region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:9-12 or 69-71, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs:9-12 or 69-71; (d) a V_(L) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 13-18 or 72-74, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 13-18 or 72-74; (e) a V_(L) CDR2region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-24 or 75-77, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 19-24 or 75-77; and (f) a V_(L)CDR3 region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:25-30 or 78-80, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs:25-30 or 78-80.

Engineered antibodies of this disclosure include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, for the 12C5 V_(λ) region, framework region amino acidpositions 40 and 68 (using the Kabat numbering system) differ fromgermline. One or both of these positions can be backmutated to germlinesequences by making one or both of the following substitutions: L40Q andR68K.

Furthermore, for the 19A3 and the CD22.1 V_(H) regions, framework regionamino acid position 27 (using the Kabat numbering system) differs fromgermline. This position can be backmutated to the germline sequence bymaking the following substitution: R27G.

Furthermore, for the CD22.2 V_(H) region, framework region amino acidpositions 27 and 57 (using the Kabat numbering system) differs fromgermline. This position can be backmutated to the germline sequence bymaking the following substitutions: R27G and Q57N.

Furthermore, for the 16F7 V_(H) region, framework region amino acidposition 28 (using the Kabat numbering system) differs from germline.This position can be backmutated to the germline sequence by making thefollowing substitution: N28S.

Furthermore, for the 16F7 V_(K).2 region, framework region amino acidposition 85 (using the Kabat numbering system) differs from germline.This position can be backmutated to the germline sequence by making thefollowing substitution: A85T.

Furthermore, for the 23C6 V_(H) region, framework region amino acidpositions 14, 79 and 88 (using the Kabat numbering system) differ fromgermline. One, two or all three of these positions can be backmutated togermline sequences by making one, two or all three of the followingsubstitutions: T14P, V79A and A88S.

Furthermore, for the 4G6 V_(H) region, framework region amino acidpositions P, D, F, D, T, Y and F (using the Kabat numbering system)differs from germline. This position can be backmutated to the germlinesequence by making one, two, three, four, five, six or all seven of thefollowing substitution: P?A; D?G; N?S; F?Y; D?E; T?S; Y?R; F?S. NEEDINPUT RE: KABAT NUMBERING.

Furthermore, for the 4G6 V_(K)1 region framework region amino acidpositions T and D (using the Kabat numbering system) differs fromgermline. These positions can be backmutated to the germline sequence bymaking one or two of the following substitution: T?K and D?E.

Furthermore, for the 21F6 V_(H)1 region, framework region amino acidposition S and I (using the Kabat numbering system) differs fromgermline. These positions can be backmutated to the germline sequence bymaking the following substitution: S?P and I?V.

Furthermore, for the 21F6 V_(H)2 region framework region amino acidpositions S and M (using the Kabat numbering system) differs fromgermline. These positions can be backmutated to the germline sequence bymaking the following substitution: S?P and M?V.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 2003/0153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of this disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of this disclosure maybe chemically modified (e.g., one or more chemical moieties can beattached to the antibody) or be modified to alter its glycosylation,again to alter one or more functional properties of the antibody. Eachof these embodiments is described in further detail below. The numberingof residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH₂—CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG for FcγR1, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 toCo et al. Additional approaches for altering glycosylation are describedin further detail in U.S. Pat. No. 7,214,775 to Hanai et al., U.S. Pat.No. 6,737,056 to Presta, U.S. Pub No. 20070020260 to Presta, PCTPublication No. WO/2007/084926 to Dickey et al., PCT Publication No.WO/2006/089294 to Zhu et al., and PCT Publication No. WO/2007/055916 toRavetch et al., each of which is hereby incorporated by reference in itsentirety.

In one exemplary embodiment, a glycosylation site in the CDR2 region ofthe V_(H) chain of the 19A3 antibody was eliminated by introducing anN57Q mutation (see Example 1), to give the recombinant antibody CD22.2having the V_(H) amino acid sequence shown in SEQ ID NO:61.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of this disclosure to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 by Hanai et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanai et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). Antibodies with a modified glycosylation profile canalso be produced in chicken eggs, as described in US Patent ApplicationNo. PCT/US06/05853. Alternatively, antibodies with a modifiedglycosylation profile can be produced in plant cells, such as Lemna.Methods for production of antibodies in a plant system are disclosed inthe U.S. patent application corresponding to Alston & Bird LLP attorneydocket No. 040989/314911, filed on Aug. 11, 2006. PCT Publication WO99/54342 by Umana et al. describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidasealpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino,A. L. et al. (1975) Biochem. 14:5516-23).

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, wherein that alteration relates to thelevel of sialyation of the antibody. Such alterations are described inPCT Publication No. WO/2007/084926 to Dickey et al, and PCT PublicationNo. WO/2007/055916 to Ravetch et al., both of which are incorporated byreference in their entirety. For example, one may employ an enzymaticreaction with sialidase, such as, for example, Arthrobacter ureafacenssialidase. The conditions of such a reaction are generally described inthe U.S. Pat. No. 5,831,077, which is hereby incorporated by referencein its entirety. Other non-limiting examples of suitable enzymes areneuraminidase and N-Glycosidase F, as described in Schloemer et al., J.Virology, 15(4), 882-893 (1975) and in Leibiger et al., Biochem J., 338,529-538 (1999), respectively. Desialylated antibodies may be furtherpurified by using affinity chromatography. Alternatively, one may employmethods to increase the level of sialyation, such as by employingsialyltransferase enzymes. Conditions of such a reaction are generallydescribed in Basset et al., Scandinavian Journal of Immunology, 51(3),307-311 (2000).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of this disclosure. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Antibody Fragments and Antibody Mimetics

The instant invention is not limited to traditional antibodies and maybe practiced through the use of antibody fragments and antibodymimetics. As detailed below, a wide variety of antibody fragment andantibody mimetic technologies have now been developed and are widelyknown in the art. While a number of these technologies, such as domainantibodies, Nanobodies, and UniBodies make use of fragments of, or othermodifications to, traditional antibody structures, there are alsoalternative technologies, such as Affibodies, DARPins, Anticalins,Avimers, and Versabodies that employ binding structures that, while theymimic traditional antibody binding, are generated from and function viadistinct mechanisms.

Domain Antibodies (dAbs) are the smallest functional binding units ofantibodies, corresponding to the variable regions of either the heavy(VH) or light (VL) chains of human antibodies. Domain Antibodies have amolecular weight of approximately 13 kDa. Domantis has developed aseries of large and highly functional libraries of fully human VH and VLdAbs (more than ten billion different sequences in each library), anduses these libraries to select dAbs that are specific to therapeutictargets. In contrast to many conventional antibodies, Domain Antibodiesare well expressed in bacterial, yeast, and mammalian cell systems.Further details of domain antibodies and methods of production thereofmay be obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081; 6,172,197; 6,696,245; U.S. Serial No. 2004/0110941; Europeanpatent application No. 1433846 and European Patents 0368684 & 0616640;WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 andWO03/002609, each of which is herein incorporated by reference in itsentirety.

Nanobodies are antibody-derived therapeutic proteins that contain theunique structural and functional properties of naturally-occurringheavy-chain antibodies. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (CH2 and CH3).Importantly, the cloned and isolated VHH domain is a perfectly stablepolypeptide harbouring the full antigen-binding capacity of the originalheavy-chain antibody. Nanobodies have a high homology with the VHdomains of human antibodies and can be further humanized without anyloss of activity. Importantly, Nanobodies have a low immunogenicpotential, which has been confirmed in primate studies with Nanobodylead compounds.

Nanobodies combine the advantages of conventional antibodies withimportant features of small molecule drugs. Like conventionalantibodies, Nanobodies show high target specificity, high affinity fortheir target and low inherent toxicity. However, like small moleculedrugs they can inhibit enzymes and readily access receptor clefts.Furthermore, Nanobodies are extremely stable, can be administered bymeans other than injection (see, e.g., WO 04/041867, which is hereinincorporated by reference in its entirety) and are easy to manufacture.Other advantages of Nanobodies include recognizing uncommon or hiddenepitopes as a result of their small size, binding into cavities oractive sites of protein targets with high affinity and selectivity dueto their unique 3-dimensional, drug format flexibility, tailoring ofhalf-life and ease and speed of drug discovery.

Nanobodies are encoded by single genes and are efficiently produced inalmost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g.,U.S. Pat. No. 6,765,087, which is herein incorporated by reference inits entirety), molds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see,e.g., U.S. Pat. No. 6,838,254, which is herein incorporated by referencein its entirety). The production process is scalable and multi-kilogramquantities of Nanobodies have been produced. Because Nanobodies exhibita superior stability compared with conventional antibodies, they can beformulated as a long shelf-life, ready-to-use solution.

The Nanoclone method (see, e.g., WO 06/079372, which is hereinincorporated by reference in its entirety) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughout selection of B-cells and could be used in the context ofthe instant invention.

UniBodies are another antibody fragment technology, however this one isbased upon the removal of the hinge region of IgG4 antibodies. Thedeletion of the hinge region results in a molecule that is essentiallyhalf the size of traditional IgG4 antibodies and has a univalent bindingregion rather than the bivalent binding region of IgG4 antibodies. It isalso well known that IgG4 antibodies are inert and thus do not interactwith the immune system, which may be advantageous for the treatment ofdiseases where an immune response is not desired, and this advantage ispassed onto UniBodies. For example, UniBodies may function to inhibit orsilence, but not kill, the cells to which they are bound. Additionally,UniBody binding to cancer cells do not stimulate them to proliferate.Furthermore, because UniBodies are about half the size of traditionalIgG4 antibodies, they may show better distribution over larger solidtumors with potentially advantageous efficacy. UniBodies are clearedfrom the body at a similar rate to whole IgG4 antibodies and are able tobind with a similar affinity for their antigens as whole antibodies.Further details of UniBodies may be obtained by reference to patentapplication WO2007/059782, which is herein incorporated by reference inits entirety.

Affibody molecules represent a new class of affinity proteins based on a58-amino acid residue protein domain, derived from one of theIgG-binding domains of staphylococcal protein A. This three helix bundledomain has been used as a scaffold for the construction of combinatorialphagemid libraries, from which Affibody variants that target the desiredmolecules can be selected using phage display technology (Nord K,Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P A, Bindingproteins selected from combinatorial libraries of an α-helical bacterialreceptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H,Uhlen M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands fromcombinatorial engineering of protein A, Eur J Biochem 2002;269:2647-55). The simple, robust structure of Affibody molecules incombination with their low molecular weight (6 kDa), make them suitablefor a wide variety of applications, for instance, as detection reagents(Ronmark J, Hansson M, Nguyen T, et al, Construction andcharacterization of affibody-Fc chimeras produced in Escherichia coli, JImmunol Methods 2002; 261:199-211) and to inhibit receptor interactions(Sandstorm K, Xu Z, Forsberg G, Nygren P A, Inhibition of the CD28-CD80co-stimulation signal by a CD28-binding Affibody ligand developed bycombinatorial protein engineering, Protein Eng 2003; 16:691-7). Furtherdetails of Affibodies and methods of production thereof may be obtainedby reference to U.S. Pat. No. 5,831,012 which is herein incorporated byreference in its entirety.

Labelled Affibodies may also be useful in imaging applications fordetermining abundance of Isoforms.

DARPins (Designed Ankyrin Repeat Proteins) are one example of anantibody mimetic DRP (Designed Repeat Protein) technology that has beendeveloped to exploit the binding abilities of non-antibody polypeptides.Repeat proteins such as ankyrin or leucine-rich repeat proteins, areubiquitous binding molecules, which occur, unlike antibodies, intra- andextracellularly. Their unique modular architecture features repeatingstructural units (repeats), which stack together to form elongatedrepeat domains displaying variable and modular target-binding surfaces.Based on this modularity, combinatorial libraries of polypeptides withhighly diversified binding specificities can be generated. This strategyincludes the consensus design of self-compatible repeats displayingvariable surface residues and their random assembly into repeat domains.

DARPins can be produced in bacterial expression systems at very highyields and they belong to the most stable proteins known. Highlyspecific, high-affinity DARPins to a broad range of target proteins,including human receptors, cytokines, kinases, human proteases, virusesand membrane proteins, have been selected. DARPins having affinities inthe single-digit nanomolar to picomolar range can be obtained.

DARPins have been used in a wide range of applications, including ELISA,sandwich ELISA, flow cytometric analysis (FACS), immunohistochemistry(IHC), chip applications, affinity purification or Western blotting.DARPins also proved to be highly active in the intracellular compartmentfor example as intracellular marker proteins fused to green fluorescentprotein (GFP). DARPins were further used to inhibit viral entry withIC50 in the pM range. DARPins are not only ideal to blockprotein-protein interactions, but also to inhibit enzymes. Proteases,kinases and transporters have been successfully inhibited, most often anallosteric inhibition mode. Very fast and specific enrichments on thetumor and very favorable tumor to blood ratios make DARPins well suitedfor in vivo diagnostics or therapeutic approaches.

Additional information regarding DARPins and other DRP technologies canbe found in U.S. Patent Application Publication No. 2004/0132028 andInternational Patent Application Publication No. WO 02/20565, both ofwhich are hereby incorporated by reference in their entirety.

Anticalins are an additional antibody mimetic technology, however inthis case the binding specificity is derived from lipocalins, a familyof low molecular weight proteins that are naturally and abundantlyexpressed in human tissues and body fluids. Lipocalins have evolved toperform a range of functions in vivo associated with the physiologicaltransport and storage of chemically sensitive or insoluble compounds.Lipocalins have a robust intrinsic structure comprising a highlyconserved β-barrel which supports four loops at one terminus of theprotein. These loops form the entrance to a binding pocket andconformational differences in this part of the molecule account for thevariation in binding specificity between individual lipocalins.

While the overall structure of hypervariable loops supported by aconserved β-sheet framework is reminiscent of immunoglobulins,lipocalins differ considerably from antibodies in terms of size, beingcomposed of a single polypeptide chain of 160-180 amino acids which ismarginally larger than a single immunoglobulin domain.

Lipocalins are cloned and their loops are subjected to engineering inorder to create Anticalins. Libraries of structurally diverse Anticalinshave been generated and Anticalin display allows the selection andscreening of binding function, followed by the expression and productionof soluble protein for further analysis in prokaryotic or eukaryoticsystems. Studies have successfully demonstrated that Anticalins can bedeveloped that are specific for virtually any human target protein canbe isolated and binding affinities in the nanomolar or higher range canbe obtained.

Anticalins can also be formatted as dual targeting proteins, so-calledDuocalins. A Duocalin binds two separate therapeutic targets in oneeasily produced monomeric protein using standard manufacturing processeswhile retaining target specificity and affinity regardless of thestructural orientation of its two binding domains.

Modulation of multiple targets through a single molecule is particularlyadvantageous in diseases known to involve more than a single causativefactor. Moreover, bi- or multivalent binding formats such as Duocalinshave significant potential in targeting cell surface molecules indisease, mediating agonistic effects on signal transduction pathways orinducing enhanced internalization effects via binding and clustering ofcell surface receptors. Furthermore, the high intrinsic stability ofDuocalins is comparable to monomeric Anticalins, offering flexibleformulation and delivery potential for Duocalins.

Additional information regarding Anticalins can be found in U.S. Pat.No. 7,250,297 and International Patent Application Publication No. WO99/16873, both of which are hereby incorporated by reference in theirentirety.

Another antibody mimetic technology useful in the context of the instantinvention are Avimers. Avimers are evolved from a large family of humanextracellular receptor domains by in vitro exon shuffling and phagedisplay, generating multidomain proteins with binding and inhibitoryproperties. Linking multiple independent binding domains has been shownto create avidity and results in improved affinity and specificitycompared with conventional single-epitope binding proteins. Otherpotential advantages include simple and efficient production ofmultitarget-specific molecules in Escherichia coli, improvedthermostability and resistance to proteases. Avimers with sub-nanomolaraffinities have been obtained against a variety of targets.

Additional information regarding Avimers can be found in U.S. PatentApplication Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114,2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932,2005/0053973, 2005/0048512, 2004/0175756, all of which are herebyincorporated by reference in their entirety.

Versabodies are another antibody mimetic technology that could be usedin the context of the instant invention. Versabodies are small proteinsof 3-5 kDa with >15% cysteines, which form a high disulfide densityscaffold, replacing the hydrophobic core that typical proteins have. Thereplacement of a large number of hydrophobic amino acids, comprising thehydrophobic core, with a small number of disulfides results in a proteinthat is smaller, more hydrophilic (less aggregation and non-specificbinding), more resistant to proteases and heat, and has a lower densityof T-cell epitopes, because the residues that contribute most to MHCpresentation are hydrophobic. All four of these properties arewell-known to affect immunogenicity, and together they are expected tocause a large decrease in immunogenicity.

The inspiration for Versabodies comes from the natural injectablebiopharmaceuticals produced by leeches, snakes, spiders, scorpions,snails, and anemones, which are known to exhibit unexpectedly lowimmunogenicity. Starting with selected natural protein families, bydesign and by screening the size, hydrophobicity, proteolytic antigenprocessing, and epitope density are minimized to levels far below theaverage for natural injectable proteins.

Given the structure of Versabodies, these antibody mimetics offer aversatile format that includes multi-valency, multi-specificity, adiversity of half-life mechanisms, tissue targeting modules and theabsence of the antibody Fc region. Furthermore, Versabodies aremanufactured in E. coli at high yields, and because of theirhydrophilicity and small size, Versabodies are highly soluble and can beformulated to high concentrations. Versabodies are exceptionally heatstable (they can be boiled) and offer extended shelf-life.

Additional information regarding Versabodies can be found in U.S. PatentApplication Publication No. 2007/0191272 which is hereby incorporated byreference in its entirety.

The detailed description of antibody fragment and antibody mimetictechnologies provided above is not intended to be a comprehensive listof all technologies that could be used in the context of the instantspecification. For example, and also not by way of limitation, a varietyof additional technologies including alternative polypeptide-basedtechnologies, such as fusions of complimentary determining regions asoutlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007),which is hereby incorporated by reference in its entirety, as well asnucleic acid-based technologies, such as the RNA aptamer technologiesdescribed in U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566,6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and 6,387,620,all of which are hereby incorporated by reference, could be used in thecontext of the instant invention.

Antibody Physical Properties

The antibodies of the present disclosure may be further characterized bythe various physical properties of the anti-CD22 antibodies. Variousassays may be used to detect and/or differentiate different classes ofantibodies based on these physical properties.

In some embodiments, antibodies of the present disclosure may containone or more glycosylation sites in either the light or heavy chainvariable region. The presence of one or more glycosylation sites in thevariable region may result in increased immunogenicity of the antibodyor an alteration of the pK of the antibody due to altered antigenbinding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala F A andMorrison S L (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro R G (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. Variable region glycosylation may be tested using a Glycoblotassay, which cleaves the antibody to produce a Fab, and then tests forglycosylation using an assay that measures periodate oxidation andSchiff base formation. Alternatively, variable region glycosylation maybe tested using Dionex light chromatography (Dionex-LC), which cleavessaccharides from a Fab into monosaccharides and analyzes the individualsaccharide content. In some instances, it is preferred to have ananti-CD22 antibody that does not contain variable region glycosylation.This can be achieved either by selecting antibodies that do not containthe glycosylation motif in the variable region or by mutating residueswithin the glycosylation motif using standard techniques well known inthe art.

In a preferred embodiment, the antibodies of the present disclosure donot contain asparagine isomerism sites. A deamidation or isoasparticacid effect may occur on N-G or D-G sequences, respectively. Thedeamidation or isoaspartic acid effect results in the creation ofisoaspartic acid which decreases the stability of an antibody bycreating a kinked structure off a side chain carboxy terminus ratherthan the main chain. The creation of isoaspartic acid can be measuredusing an iso-quant assay, which uses a reverse-phase HPLC to test forisoaspartic acid.

Each antibody will have a unique isoelectric point (pI), but generallyantibodies will fall in the pH range of between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.Antibodies may have a pI that is outside this range. Although theeffects are generally unknown, there is speculation that antibodies witha pI outside the normal range may have some unfolding and instabilityunder in vivo conditions. The isoelectric point may be tested using acapillary isoelectric focusing assay, which creates a pH gradient andmay utilize laser focusing for increased accuracy (Janini et al (2002)Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89;Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it ispreferred to have an anti-CD22 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range, or by mutating charged surfaceresidues using standard techniques well known in the art.

Each antibody will have a melting temperature that is indicative ofthermal stability (Krishnamurthy R and Manning M C (2002) Curr PharmBiotechnol 3:361-71). A higher thermal stability indicates greateroverall antibody stability in vivo. The melting point of an antibody maybe measure using techniques such as differential scanning calorimetry(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLett 68:47-52). T_(M1) indicates the temperature of the initialunfolding of the antibody. T_(M2) indicates the temperature of completeunfolding of the antibody. Generally, it is preferred that the T_(M1) ofan antibody of the present disclosure is greater than 60° C., preferablygreater than 65° C., even more preferably greater than 70° C.Alternatively, the thermal stability of an antibody may be measure usingcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not rapidlydegrade. Fragmentation of an anti-CD22 antibody may be measured usingcapillary electrophoresis (CE) and MALDI-MS, as is well understood inthe art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects. Aggregation may lead to triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation may be measured by several techniques well known in the art,including size-exclusion column (SEC) high performance liquidchromatography (HPLC), and light scattering to identify monomers,dimers, trimers or multimers.

Methods of Engineering Antibodies

As discussed above, the anti-CD22 antibodies having V_(H) and V_(L)sequences disclosed herein can be used to create new anti-CD22antibodies by modifying the V_(H) and/or V_(L) sequences, or theconstant region(s) attached thereto. Thus, in another aspect of thisdisclosure, the structural features of an anti-CD22 antibody of thisdisclosure, e.g. 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6,are used to create structurally related anti-CD22 antibodies that retainat least one functional property of the antibodies of this disclosure,such as binding to human CD22. For example, one or more CDR regions of12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6, or mutationsthereof, can be combined recombinantly with known framework regionsand/or other CDRs to create additional, recombinantly-engineered,anti-CD22 antibodies of this disclosure, as discussed above. Other typesof modifications include those described in the previous section. Thestarting material for the engineering method is one or more of the V_(H)and/or V_(L) sequences provided herein, or one or more CDR regionsthereof. To create the engineered antibody, it is not necessary toactually prepare (i.e., express as a protein) an antibody having one ormore of the V_(H) and/or V_(L) sequences provided herein, or one or moreCDR regions thereof. Rather, the information contained in thesequence(s) is used as the starting material to create a “secondgeneration” sequence(s) derived from the original sequence(s) and thenthe “second generation” sequence(s) is prepared and expressed as aprotein.

Accordingly, in another embodiment, this disclosure provides a methodfor preparing an anti-CD22 antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 1-4 and 63-65; a CDR2 sequence selected from the group consistingof SEQ ID NOs: 5-8, 60, and 66-68 and/or a CDR3 sequence selected fromthe group consisting of SEQ ID NOs: 9-12 and 69-71; and/or (ii) a lightchain variable region antibody sequence comprising a CDR1 sequenceselected from the group consisting of SEQ ID NOs: 13-18 and 72-74; aCDR2 sequence selected from the group consisting of SEQ ID NOs: 19-24and 75-77; and/or a CDR3 sequence selected from the group consisting ofSEQ ID NOs: 25-30 and 78-80;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

For example, standard molecular biology techniques can be used toprepare and express the altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-CD22 antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (a) internalizing into CD22+ cells;    -   (b) exhibiting ADCC activity on CD22+ cells;    -   (c) enhancing cell death of Ramos cells induced by BCR        stimulation;    -   (d) not having a direct anti-proliferative effect on Ramos        cells;    -   (d) not inducing calcium flux in Ramos cells;    -   (e) not mediating CDC activity on Ramos cells; and/or    -   (f) inhibits growth of CD22-expressing cells in vivo when        conjugated to a cytotoxin

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples.

In certain embodiments of the methods of engineering antibodies of thisdisclosure, mutations can be introduced randomly or selectively alongall or part of an anti-CD22 antibody coding sequence and the resultingmodified anti-CD22 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of this Disclosure

Another aspect of this disclosure pertains to nucleic acid moleculesthat encode the antibodies of this disclosure. The nucleic acids may bepresent in whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of this disclosure can be, for example, DNA or RNA and mayor may not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of this disclosure can be obtained using standardmolecular biology techniques. For antibodies expressed by hybridomas(e.g., hybridomas prepared from transgenic mice carrying humanimmunoglobulin genes as described further below), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), a nucleic acid encoding such antibodies canbe recovered from the gene library.

Preferred nucleic acids molecules of this disclosure are those encodingthe V_(H) and V_(L) sequences of the 12C5, 19A3, CD22.1, CD22.2, 16F7,23C6, 4G6 and 21F6 monoclonal antibodies. DNA sequences encoding theV_(H) sequences of 12C5, 19A3, CD22.1, 16F7, 23C6, CD22.2, 4G6 and 21F6are shown in SEQ ID NOs: 41-44, 62 and 87-89, respectively (wherein theheavy chains of 19A3 and CD22.1 are identical and correspond to SEQ IDNO:42; the heavy chain of CD22.2 corresponds to SEQ ID NO:62; and theheavy chains of 21F6 correspond to SEQ ID NOs:82 and 83). DNA sequencesencoding the V_(L) sequences of 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6,4G6 and 21F6 are shown in SEQ ID NOs: 45-50 and 90-92, respectively(wherein the kappa light chains of 19A3, CD22.1 and CD22.2 are identicaland correspond to SEQ ID NO:46, the kappa light chain of 16F7corresponds to either SEQ ID NO:47 or 48, the kappa light chain of 23C6corresponds to either SEQ ID NO:49 or 50, and the kappa light chain of4G6 corresponds to either SEQ ID NO:90 or 91).

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., el al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgQ1,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. In preferred embodiments,the light chain constant region can be a kappa or lambda constantregion.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Monoclonal Antibodies of this Disclosure

Monoclonal antibodies (mAbs) of the present disclosure can be producedby a variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

In a preferred embodiment, the antibodies of this disclosure are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstCD22 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asthe HuMAb Mouse® and KM Mouse®, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, andHarding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546).Preparation and use of the HuMAb Mouse®, and the genomic modificationscarried by such mice, is further described in Taylor, L. et al. (1992)Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl.Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.(1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; and Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851, the contents of all of which are herebyspecifically incorporated by reference in their entirety. See further,U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all toLonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of this disclosure can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. This mouse isreferred to herein as a “KM Mouse®,” and is described in detail in PCTPublication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD22 antibodies of this disclosure. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD22 antibodies of this disclosure. For example, mice carrying botha human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (e.g., Kuroiwa et al.(2002) Nature Biotechnology 20:889-894 and PCT application No. WO2002/092812) and can be used to raise anti-CD22 antibodies of thisdisclosure.

Human monoclonal antibodies of this disclosure can also be preparedusing phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art. See for example: U.S. Pat. Nos.5,223,409; 5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S.Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos.5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

In another embodiment, human anti-CD22 antibodies are prepared using acombination of human Ig mouse and phage display techniques, as describedin U.S. Pat. No. 6,794,132 by Buechler et al. More specifically, themethod first involves raising an anti-CD22 antibody response in a humanIg mouse (such as a HuMab mouse or KM mouse as described above) byimmunizing the mouse with a CD22 antigen, followed by isolating nucleicacids encoding human antibody chains from lymphatic cells of the mouseand introducing these nucleic acids into a display vector (e.g., phage)to provide a library of display packages. Thus, each library membercomprises a nucleic acid encoding a human antibody chain and eachantibody chain is displayed from the display package. The library thenis screened with a CD22 antigen to isolate library members thatspecifically bind CD22. Nucleic acid inserts of the selected librarymembers then are isolated and sequenced by standard methods to determinethe light and heavy chain variable sequences of the selected CD22binders. The variable regions can be converted to full-length antibodychains by standard recombinant DNA techniques, such as cloning of thevariable regions into an expression vector that carries the human heavyand light chain constant regions such that the V_(H) region isoperatively linked to the C_(H) region and the V_(L) region isoperatively linked to the C_(L) region.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of thisdisclosure, such mice can be immunized with a purified or enrichedpreparation of CD22 antigen and/or recombinant CD22, or cells expressingCD22, or a CD22 fusion protein, as described by Lonberg, N. et al.(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or recombinant preparation (5-50 μg)of CD22 antigen can be used to immunize the human Ig miceintraperitoneally. Most preferably, the immunogen used to raise theantibodies of this disclosure is a combination of recombinant human CD22extracellular domain and CHO cells engineered to express full-lengthhuman CD22 on the cell surface (described further in Example 1).

Detailed procedures to generate fully human monoclonal antibodies toCD22 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-CD22 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12). Alternatively or additionally, the KMMouse® and/or KM-λHAC strains can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of thisDisclosure

To generate hybridomas producing human monoclonal antibodies of thisdisclosure, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Alternatively, the single cell suspensionof splenic lymphocytes from immunized mice can be fused using anelectric field based electrofusion method, using a CytoPulse largechamber cell fusion electroporator (CytoPulse Sciences, Inc., GlenBurnie Md.). Cells are plated at approximately 2×10⁵ in flat bottommicrotiter plate, followed by a two week incubation in selective mediumcontaining 20% fetal Clone Serum, 18% “653” conditioned media, 5% origen(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After approximately two weeks, cells can be cultured in mediumin which the HAT is replaced with HT. Individual wells can then bescreened by ELISA for human monoclonal IgM and IgG antibodies. Onceextensive hybridoma growth occurs, medium can be observed usually after10-14 days. The antibody secreting hybridomas can be replated, screenedagain, and if still positive for human IgG, the monoclonal antibodiescan be subcloned at least twice by limiting dilution. The stablesubclones can then be cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of thisDisclosure

Antibodies of this disclosure also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can beinserted into separate vector or, more typically, both genes areinserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector and the V_(L) segment is operatively linkedto the C_(L) segment within the vector. Additionally or alternatively,the recombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of this disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of this disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all to Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr− host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of this disclosure in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof this disclosure include Chinese Hamster Ovary (CHO cells) (includingdhfr⁻ CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462 (to Wilson), WO89/01036 (to Bebbington) and EP 338,841 (to Bebbington). Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to CD22 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified and/or recombinant CD22 (e.g., CD22 ECD as described inExample 1) at 0.25 μg/ml in PBS, and then blocked with 5% bovine serumalbumin in PBS. Dilutions of antibody (e.g., dilutions of plasma fromCD22-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to alkaline phosphatase for 1hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micethat develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with CD22 immunogen. Hybridomasthat bind with high avidity to CD22 are subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-CD22 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-CD22 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using CD22 coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-CD22 human IgGs can be further tested for reactivity with CD22antigen by Western blotting. Briefly, CD22 can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

The binding specificity of an antibody of this disclosure may also bedetermined by monitoring binding of the antibody to cells expressingCD22, for example by flow cytometry. A cell line that naturallyexpresses CD22, such as Daudi cells or Raji cells, may be used or a cellline, such as a CHO cell line, may be transfected with an expressionvector encoding a transmembrane form of CD22. The transfected proteinmay comprise a tag, such as a myc-tag, preferably at the N-terminus, fordetection using an antibody to the tag. Binding of an antibody of thisdisclosure to CD22 may be determined by incubating the transfected cellswith the antibody, and detecting bound antibody. Binding of an antibodyto the tag on the transfected protein may be used as a positive control.

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising an anti-CD22 antibody, or a fragment thereof, of thisdisclosure. An antibody of this disclosure, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of thisdisclosure may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthis disclosure, an antibody of this disclosure can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for CD22 and a secondbinding specificity for a second target epitope. In a particularembodiment of this disclosure, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, this disclosure includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing CD22. These bispecific molecules target CD22 expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of CD22 expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

In an embodiment of this disclosure in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-CD22 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g. viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

In one embodiment, the bispecific molecules of this disclosure compriseas a binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, Fd, dAb or a singlechain Fv. The antibody may also be a light chain or heavy chain dimer,or any minimal fragment thereof such as a Fv or a single chain constructas described in U.S. Pat. No. 4,946,778 to Ladner et al., the contentsof which is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγRI(CD64), FcγRII(CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹).

The production and characterization of certain preferred anti-Fcγ

antibodies are described in PCT Publication WO 88/00052 and in U.S. Pat.No. 4,954,617 to Fanger et al., the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this disclosure are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol155 (10): 4996-5002 and PCT Publication WO 94/10332 to Tempest et al.The H22 antibody producing cell line was deposited at the American TypeCulture Collection under the designation HA022CL1 and has the accessionno. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of this disclosure because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); and (4) mediate enhanced antigen presentation ofantigens, including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of this disclosure aremurine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-CD22 binding specificities, using methods known in the art. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83, and Glennie et al.(1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of thisdisclosure can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858, all of which are expresslyincorporated herein by reference.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a

counter or a scintillation counter or by autoradiography.

Linkers

The present invention provides for antibody-partner conjugates where theantibody is linked to the partner through a chemical linker. In someembodiments, the linker is a peptidyl linker, and is depicted herein as(L⁴)_(p)-F-(L¹)_(m). Other linkers include hydrazine and disulfidelinkers, and is depicted herein as (L⁴)_(p)-H-(L¹)_(m) or(L⁴)_(p)-J-(L¹)_(m), respectively. In addition to the linkers beingattached to the partner, the present invention also provides cleavablelinker arms that are appropriate for attachment to essentially anymolecular species. The linker arm aspect of the invention is exemplifiedherein by reference to their attachment to a therapeutic moiety. Itwill, however, be readily apparent to those of skill in the art that thelinkers can be attached to diverse species including, but not limitedto, diagnostic agents, analytical agents, biomolecules, targetingagents, detectable labels and the like.

The use of peptidyl and other linkers in antibody-partner conjugates isdescribed in U.S. Provisional Patent Application Ser. Nos. 60/295,196;60/295,259; 60/295,342; 60/304,908; 60/572,667; 60/661,174; 60/669,871;60/720,499; 60/730,804; 60/735,657; 60/891,028; and U.S. patentapplication Ser. Nos. 10/160,972; 10/161,234; 11/134,685; 11/134,826;and 11/398,854 and U.S. Pat. No. 6,989,452 and PCT Patent ApplicationNo. PCT/US2006/37793, all of which are incorporated herein by reference.

Additional linkers are described in U.S. Pat. No. 6,214,345(Bristol-Myers Squibb), U.S. Pat. Appl. 2003/0096743 and U.S. Pat. Appl.2003/0130189 (both to Seattle Genetics), de Groot et al., J. Med. Chem.42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093 (2000); de Grootet al., J. Med. Chem. 66, 8815, (2001); WO 02/083180 (Syntarga); Carl etal., J. Med. Chem. Lett. 24, 479, (1981); Dubowchik et al., Bioorg &Med. Chem. Lett. 8, 3347 (1998).

In one aspect, the present invention relates to linkers that are usefulto attach targeting groups to therapeutic agents and markers. In anotheraspect, the invention provides linkers that impart stability tocompounds, reduce their in vivo toxicity, or otherwise favorably affecttheir pharmacokinetics, bioavailability and/or pharmacodynamics. It isgenerally preferred that in such embodiments, the linker is cleaved,releasing the active drug, once the drug is delivered to its site ofaction. Thus, in one embodiment of the invention, the linkers of theinvention are traceless, such that once removed from the therapeuticagent or marker (such as during activation), no trace of the linker'spresence remains.

In another embodiment of the invention, the linkers are characterized bytheir ability to be cleaved at a site in or near the target cell such asat the site of therapeutic action or marker activity. Such cleavage canbe enzymatic in nature. This feature aids in reducing systemicactivation of the therapeutic agent or marker, reducing toxicity andsystemic side effects. Preferred cleavable groups for enzymatic cleavageinclude peptide bonds, ester linkages, and disulfide linkages. In otherembodiments, the linkers are sensitive to pH and are cleaved throughchanges in pH.

An important aspect of the current invention is the ability to controlthe speed with which the linkers cleave. Often a linker that cleavesquickly is desired. In some embodiments, however, a linker that cleavesmore slowly may be preferred. For example, in a sustained releaseformulation or in a formulation with both a quick release and a slowrelease component, it may be useful to provide a linker which cleavesmore slowly. WO 02/096910 provides several specific ligand-drugcomplexes having a hydrazine linker. However, there is no way to “tune”the linker composition dependent upon the rate of cyclization required,and the particular compounds described cleave the ligand from the drugat a slower rate than is preferred for many drug-linker conjugates. Incontrast, the hydrazine linkers of the current invention provide for arange of cyclization rates, from very fast to very slow, therebyallowing for the selection of a particular hydrazine linker based on thedesired rate of cyclization.

For example, very fast cyclization can be achieved with hydrazinelinkers that produce a single 5-membered ring upon cleavage. Preferredcyclization rates for targeted delivery of a cytotoxic agent to cellsare achieved using hydrazine linkers that produce, upon cleavage, eithertwo 5-membered rings or a single 6-membered ring resulting from a linkerhaving two methyls at the geminal position. The gem-dimethyl effect hasbeen shown to accelerate the rate of the cyclization reaction ascompared to a single 6-membered ring without the two methyls at thegeminal position. This results from the strain being relieved in thering. Sometimes, however, substitutents may slow down the reactioninstead of making it faster. Often the reasons for the retardation canbe traced to steric hindrance. For example, the gem dimethylsubstitution allows for a much faster cyclization reaction to occurcompared to when the geminal carbon is a CH₂.

It is important to note, however, that in some embodiments, a linkerthat cleaves more slowly may be preferred. For example, in a sustainedrelease formulation or in a formulation with both a quick release and aslow release component, it may be useful to provide a linker whichcleaves more slowly. In certain embodiments, a slow rate of cyclizationis achieved using a hydrazine linker that produces, upon cleavage,either a single 6-membered ring, without the gem-dimethyl substitution,or a single 7-membered ring.

The linkers also serve to stabilize the therapeutic agent or markeragainst degradation while in circulation. This feature provides asignificant benefit since such stabilization results in prolonging thecirculation half-life of the attached agent or marker. The linker alsoserves to attenuate the activity of the attached agent or marker so thatthe conjugate is relatively benign while in circulation and has thedesired effect, for example is toxic, after activation at the desiredsite of action. For therapeutic agent conjugates, this feature of thelinker serves to improve the therapeutic index of the agent.

The stabilizing groups are preferably selected to limit clearance andmetabolism of the therapeutic agent or marker by enzymes that may bepresent in blood or non-target tissue and are further selected to limittransport of the agent or marker into the cells. The stabilizing groupsserve to block degradation of the agent or marker and may also act inproviding other physical characteristics of the agent or marker. Thestabilizing group may also improve the agent or marker's stabilityduring storage in either a formulated or non-formulated form.

Ideally, the stabilizing group is useful to stabilize a therapeuticagent or marker if it serves to protect the agent or marker fromdegradation when tested by storage of the agent or marker in human bloodat 37° C. for 2 hours and results in less than 20%, preferably less than10%, more preferably less than 5% and even more preferably less than 2%,cleavage of the agent or marker by the enzymes present in the humanblood under the given assay conditions.

The present invention also relates to conjugates containing theselinkers. More particularly, the invention relates to prodrugs that maybe used for the treatment of disease, especially for cancerchemotherapy. Specifically, use of the linkers described herein providefor prodrugs that display a high specificity of action, a reducedtoxicity, and an improved stability in blood relative to prodrugs ofsimilar structure.

The linkers of the present invention as described herein may be presentat a variety of positions within the partner molecule.

Thus, there is provided a linker that may contain any of a variety ofgroups as part of its chain that will cleave in vivo, e.g., in the bloodstream, at a rate which is enhanced relative to that of constructs thatlack such groups. Also provided are conjugates of the linker arms withtherapeutic and diagnostic agents. The linkers are useful to formprodrug analogs of therapeutic agents and to reversibly link atherapeutic or diagnostic agent to a targeting agent, a detectablelabel, or a solid support. The linkers may be incorporated intocomplexes that include the cytotoxins of the invention.

In addition to the cleavable peptide, hydrazine, or disulfide group, oneor more self-immolative linker groups L¹ are optionally introducedbetween the cytoCytotoxin And the targeting agent. These linker groupsmay also be described as spacer groups and contain at least two reactivefunctional groups. Typically, one chemical functionality of the spacergroup bonds to a chemical functionality of the therapeutic agent, e.g.,cytotoxin, while the other chemical functionality of the spacer group isused to bond to a chemical functionality of the targeting agent or thecleavable linker. Examples of chemical functionalities of spacer groupsinclude hydroxy, mercapto, carbonyl, carboxy, amino, ketone, andmercapto groups.

The self-immolative linkers, represented by L¹, are generally asubstituted or unsubstituted alkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl or substituted or unsubstitutedheteroalkyl group. In one embodiment, the alkyl or aryl groups maycomprise between 1 and 20 carbon atoms. They may also comprise apolyethylene glycol moiety.

Exemplary spacer groups include, for example, 6-aminohexanol,6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other aminoacids, 1,6-hexanediol, β-alanine, 2-aminoethanol, cysteamine(2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid,3-maleimidobenzoic acid, phthalide, α-substituted phthalides, thecarbonyl group, aminal esters, nucleic acids, peptides and the like.

The spacer can serve to introduce additional molecular mass and chemicalfunctionality into the cytotoxin-targeting agent complex. Generally, theadditional mass and functionality will affect the serum half-life andother properties of the complex. Thus, through careful selection ofspacer groups, cytotoxin complexes with a range of serum half-lives canbe produced.

The spacer(s) located directly adjacent to the drug moiety is alsodenoted as (L¹)_(m), wherein m is an integer selected from 0, 1, 2, 3,4, 5, and 6. When multiple L¹ spacers are present, either identical ordifferent spacers may be used. L¹ may be any self-immolative group.

L⁴ is a linker moiety that preferably imparts increased solubility ordecreased aggregation properties to conjugates utilizing a linker thatcontains the moiety or modifies the hydrolysis rate of the conjugate.The L⁴ linker does not have to be self immolative. In one embodiment,the L⁴ moiety is substituted alkyl, unsubstituted alkyl, substitutedaryl, unsubstituted aryl, substituted heteroalkyl, or unsubstitutedheteroalkyl, any of which may be straight, branched, or cyclic. Thesubstitutions may be, for example, a lower (C¹-C⁶) alkyl, alkoxy,aklylthio, alkylamino, or dialkylamino. In certain embodiments, L⁴comprises a non-cyclic moiety. In another embodiment, L⁴ comprises anypositively or negatively charged amino acid polymer, such as polylysineor polyargenine. L⁴ can comprise a polymer such as a polyethylene glycolmoiety. Additionally the L⁴ linker can comprise, for example, both apolymer component and a small chemical moiety.

In a preferred embodiment, L⁴ comprises a polyethylene glycol (PEG)moiety. The PEG portion of L⁴ may be between 1 and 50 units long.Preferably, the PEG will have 1-12 repeat units, more preferably 3-12repeat units, more preferably 2-6 repeat units, or even more preferably3-5 repeat units and most preferably 4 repeat units. L⁴ may consistsolely of the PEG moiety, or it may also contain an additionalsubstituted or unsubstituted alkyl or heteroalkyl. It is useful tocombine PEG as part of the L⁴ moiety to enhance the water solubility ofthe complex. Additionally, the PEG moiety reduces the degree ofaggregation that may occur during the conjugation of the drug to theantibody.

In some embodiments, L⁴ comprises

directly attached to the N-terminus of (AA¹)_(c). R²⁰ is a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, and acyl. Each R²⁵, R^(25′), R²⁶, and R^(26′)is independently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocycloalkyl; and s and t are independently integersfrom 1 to 6. Preferably, R²⁰, R²⁵, R^(25′), R²⁶ and R^(26′) arehydrophobic. In some embodiments, R²⁰ is H or alkyl (preferably,unsubstituted lower alkyl). In some embodiments, R²⁵, R^(25′), R²⁶ andR^(26′) are independently H or alkyl (preferably, unsubstituted C¹ to C⁴alkyl). In some embodiments, R²⁵, R^(25′), R²⁶ and R^(26′) are all H. Insome embodiments, t is 1 and s is 1 or 2.

Peptide Linkers (F)

As discussed above, the peptidyl linkers of the invention can berepresented by the general formula: (L⁴)_(p)-F-(L¹)_(m), wherein Frepresents the linker portion comprising the peptidyl moiety. In oneembodiment, the F portion comprises an optional additionalself-immolative linker(s), L², and a carbonyl group. In anotherembodiment, the F portion comprises an amino group and an optionalspacer group(s), L³.

Accordingly, in one embodiment, the conjugate comprising the peptidyllinker comprises a structure of the following formula (a):

In this embodiment, L¹ is a self-immolative linker, as described above,and L⁴ is a moiety that preferably imparts increased solubility, ordecreased aggregation properties, or modifies the hydrolysis rate, asdescribed above. L² represents a self-immolative linker(s). In addition,m is 0, 1, 2, 3, 4, 5, or 6; and o and p are independently 0 or 1. AA¹represents one or more natural amino acids, and/or unnatural α-aminoacids; c is an integer from 1 and 20. In some embodiments, c is in therange of 2 to 5 or c is 2 or 3.

In the peptide linkers of the invention of the above formula (a), AA¹ islinked, at its amino terminus, either directly to L⁴ or, when L⁴ isabsent, directly to the X⁴ group (i.e., the targeting agent, detectablelabel, protected reactive functional group or unprotected reactivefunctional group). In some embodiments, when L⁴ is present, L⁴ does notcomprise a carboxylic acyl group directly attached to the N-terminus of(AA¹)_(c). Thus, it is not necessary in these embodiments for there tobe a carboxylic acyl unit directly between either L⁴ or X⁴ and AA¹, asis necessary in the peptidic linkers of U.S. Pat. No. 6,214,345.

In another embodiment, the conjugate comprising the peptidyl linkercomprises a structure of the following formula (b):

In this embodiment, L⁴ is a moiety that preferably imparts increasedsolubility, or decreased aggregation properties, or modifies thehydrolysis rate, as described above; L³ is a spacer group comprising aprimary or secondary amine or a carboxyl functional group, and eitherthe amine of L³ forms an amide bond with a pendant carboxyl functionalgroup of D or the carboxyl of L³ forms an amide bond with a pendantamine functional group of D; and o and p are independently 0 or 1. AA¹represents one or more natural amino acids, and/or unnatural α-aminoacids; c is an integer from 1 and 20. In this embodiment, L¹ is absent(i.e., m is 0 in the general formula).

In the peptide linkers of the invention of the above formula (b), AA¹ islinked, at its amino terminus, either directly to L⁴ or, when L⁴ isabsent, directly to the X⁴ group (i.e., the targeting agent, detectablelabel, protected reactive functional group or unprotected reactivefunctional group). In some embodiments, when L⁴ is present, L⁴ does notcomprise a carboxylic acyl group directly attached to the N-terminus of(AA¹)_(c). Thus, it is not necessary in these embodiments for there tobe a carboxylic acyl unit directly between either L⁴ or X⁴ and AA¹, asis necessary in the peptidic linkers of U.S. Pat. No. 6,214,345.

The Self-Immolative Linker L²

The self-immolative linker L² is a bifunctional chemical moiety which iscapable of covalently linking together two spaced chemical moieties intoa normally stable tripartate molecule, releasing one of said spacedchemical moieties from the tripartate molecule by means of enzymaticcleavage; and following said enzymatic cleavage, spontaneously cleavingfrom the remainder of the molecule to release the other of said spacedchemical moieties. In accordance with the present invention, theself-immolative spacer is covalently linked at one of its ends to thepeptide moiety and covalently linked at its other end to the chemicallyreactive site of the drug moiety whose derivatization inhibitspharmacological activity, so as to space and covalently link togetherthe peptide moiety and the drug moiety into a tripartate molecule whichis stable and pharmacologically inactive in the absence of the targetenzyme, but which is enzymatically cleavable by such target enzyme atthe bond covalently linking the spacer moiety and the peptide moiety tothereby effect release of the peptide moiety from the tripartatemolecule. Such enzymatic cleavage, in turn, will activate theself-immolating character of the spacer moiety and initiate spontaneouscleavage of the bond covalently linking the spacer moiety to the drugmoiety, to thereby effect release of the drug in pharmacologicallyactive form.

The self-immolative linker L² may be any self-immolative group.Preferably L² is a substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, unsubstituted heterocycloalkyl,substituted heterocycloalkyl, substituted and unsubstituted aryl, andsubstituted and unsubstituted heteroaryl.

One particularly preferred self-immolative spacer L² may be representedby the formula (c):

The aromatic ring of the aminobenzyl group may be substituted with oneor more “K” groups. A “K” group is a substituent on the aromatic ringthat replaces a hydrogen otherwise attached to one of the fournon-substituted carbons that are part of the ring structure. The “K”group may be a single atom, such as a halogen, or may be a multi-atomgroup, such as alkyl, heteroalkyl, amino, nitro, hydroxy, alkoxy,haloalkyl, and cyano. Each K is independently selected from the groupconsisting of substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedheterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO₂, NR²¹R²²,NR²¹COR²², OCONR²¹R²², OCOR²¹, and OR²¹, wherein R²¹ and R²² areindependently selected from the group consisting of H, substitutedalkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstitutedheteroalkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl andunsubstituted heterocycloalkyl. Exemplary K substituents include, butare not limited to, F, Cl, Br, I, NO₂, OH, OCH₃, NHCOCH₃, N(CH₃)₂,NHCOCF₃ and methyl. For “K_(i)”, i is an integer of 0, 1, 2, 3, or 4. Inone preferred embodiment, i is 0.

The ether oxygen atom of the structure shown above is connected to acarbonyl group. The line from the NR²⁴ functionality into the aromaticring indicates that the amine functionality may be bonded to any of thefive carbons that both form the ring and are not substituted by the—CH₂—O— group. Preferably, the NR²⁴ functionality of X is covalentlybound to the aromatic ring at the para position relative to the —CH₂—O—group. R²⁴ is a member selected from the group consisting of H,substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, andunsubstituted heteroalkyl. In a specific embodiment, R²⁴ is hydrogen.

In one embodiment, the invention provides a peptide linker of formula(a) above, wherein F comprises the structure:

where R²⁴ is selected from the group consisting of H, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, and unsubstitutedheteroalkyl. Each K is a member independently selected from the groupconsisting of substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedheterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO₂, NR²¹R²²,NR²¹COR²², OCONR²¹R²², OCOR²¹, and OR²¹ where R²¹ and R²² areindependently selected from the group consisting of H, substitutedalkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstitutedheteroalkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl; and i is an integer of 0, 1, 2, 3, or 4.

In another embodiment, the peptide linker of formula (a) above comprisesa —F-(L¹)_(m)- that comprises the structure:

where each R²⁴ is a member independently selected from the groupconsisting of H substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, and unsubstituted heteroalkyl.

In some embodiments, the self-immolative spacer L¹ or L² includes

where each R¹⁷, R¹⁸, and R¹⁹ is independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl and substituted or unsubstituted aryl, and w is an integerfrom 0 to 4. In some embodiments, R¹⁷ and R¹⁸ are independently H oralkyl (preferably, unsubstituted C₁₋₄ alkyl). Preferably, R¹⁷ and R¹⁸are C1-4 alkyl, such as methyl or ethyl. In some embodiments, w is 0.While not wishing to be bound to any particular theory, it has beenfound experimentally that this particular self-immolative spacercyclizes relatively quickly.

In some embodiments, L¹ or L² includes

The Spacer Group L³

The spacer group L³ is characterized in that it comprises a primary orsecondary amine or a carboxyl functional group, and either the amine ofthe L³ group forms an amide bond with a pendant carboxyl functionalgroup of D or the carboxyl of L³ forms an amide bond with a pendantamine functional group of D. L³ can be selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedheterocycloalkyl. In a preferred embodiment, L³ comprises an aromaticgroup. More preferably, L³ comprises a benzoic acid group, an anilinegroup or indole group. Non-limiting examples of structures that canserve as an -L³-NH— spacer include the following structures:

where Z is a member selected from O, S and NR²³, and where R²³ is amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl.

Upon cleavage of the linker of the invention containing L³, the L³moiety remains attached to the drug, D. Accordingly, the L³ moiety ischosen such that its presence attached to D does not significantly alterthe activity of D. In another embodiment, a portion of the drug D itselffunctions as the L³ spacer. For example, in one embodiment, the drug, D,is a duocarmycin derivative in which a portion of the drug functions asthe L³ spacer. Non-limiting examples of such embodiments include thosein which NH₂-(L³)-D has a structure selected from the group consistingof:

where Z is a member selected from O, S and NR²³, where R²³ is a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, and acyl; and where the NH₂ group on eachstructure reacts with (AA¹)_(c) to form -(AA¹)_(c)-NH—.

The Peptide Sequence AA¹

The group AA¹ represents a single amino acid or a plurality of aminoacids that are joined together by amide bonds. The amino acids may benatural amino acids and/or unnatural α-amino acids.

The peptide sequence (AA¹)_(c) is functionally the amidification residueof a single amino acid (when c=1) or a plurality of amino acids joinedtogether by amide bonds. The peptide of the current invention isselected for directing enzyme-catalyzed cleavage of the peptide by anenzyme in a location of interest in a biological system. For example,for conjugates that are targeted to a cell using a targeting agent, butnot internalized by that cell, a peptide is chosen that is cleaved byone or more proteases that may exist in the extracellular matrix, e.g.,due to release of the cellular contents of nearby dying cells, such thatthe peptide is cleaved extracellularly. The number of amino acids withinthe peptide can range from 1 to 20; but more preferably there will be1-8 amino acids, 1-6 amino acids or 1, 2, 3 or 4 amino acids comprising(AA¹)_(c). Peptide sequences that are susceptible to cleavage byspecific enzymes or classes of enzymes are well known in the art.

Many peptide sequences that are cleaved by enzymes in the serum, liver,gut, etc. are known in the art. An exemplary peptide sequence of theinvention includes a peptide sequence that is cleaved by a protease. Thefocus of the discussion that follows on the use of a protease-sensitivesequence is for clarity of illustration and does not serve to limit thescope of the present invention.

When the enzyme that cleaves the peptide is a protease, the linkergenerally includes a peptide containing a cleavage recognition sequencefor the protease. A cleavage recognition sequence for a protease is aspecific amino acid sequence recognized by the protease duringproteolytic cleavage. Many protease cleavage sites are known in the art,and these and other cleavage sites can be included in the linker moiety.See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in Amyloid ProteinPrecursor in Development, Aging, and Alzheimer's Disease, ed. Masters etal. pp. 190-198 (1994).

The amino acids of the peptide sequence (AA¹)_(c) are chosen based ontheir suitability for selective enzymatic cleavage by particularmolecules such as tumor-associated protease. The amino acids used may benatural or unnatural amino acids. They may be in the L or the Dconfiguration. In one embodiment, at least three different amino acidsare used. In another embodiment, only two amino acids are used.

In a preferred embodiment, the peptide sequence (AA¹)_(c) is chosenbased on its ability to be cleaved by a lysosomal proteases,non-limiting examples of which include cathepsins B, C, D, H, L and S.Preferably, the peptide sequence (AA¹)_(c) is capable of being cleavedby cathepsin B in vitro, which can be tested using in vitro proteasecleavage assays known in the art.

In another embodiment, the peptide sequence (AA¹)_(c) is chosen based onits ability to be cleaved by a tumor-associated protease, such as aprotease that is found extracellularly in the vicinity of tumor cells,non-limiting examples of which include thimet oligopeptidase (TOP) andCD10. The ability of a peptide to be cleaved by TOP or CD10 can betested using in vitro protease cleavage assays known in the art.

Suitable, but non-limiting, examples of peptide sequences suitable foruse in the conjugates of the invention include Val-Cit, Cit-Cit,Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit,Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu(SEQ. ID NO: 94), β-Ala-Leu-Ala-Leu (SEQ. ID NO: 95) and Gly-Phe-Leu-Gly(SEQ. ID NO: 96), Val-Ala, Leu-Leu-Gly-Leu (SEQ. ID NO: 97),Leu-Asn-Ala, and Lys-Leu-Val. Preferred peptides sequences are Val-Citand Val-Lys.

In another embodiment, the amino acid located the closest to the drugmoiety is selected from the group consisting of: Ala, Asn, Asp, Cit,Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,and Val. In yet another embodiment, the amino acid located the closestto the drug moiety is selected from the group consisting of: Ala, Asn,Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr,and Val.

Proteases have been implicated in cancer metastasis. Increased synthesisof the protease urokinase was correlated with an increased ability tometastasize in many cancers. Urokinase activates plasmin fromplasminogen, which is ubiquitously located in the extracellular spaceand its activation can cause the degradation of the proteins in theextracellular matrix through which the metastasizing tumor cells invade.Plasmin can also activate the collagenases thus promoting thedegradation of the collagen in the basement membrane surrounding thecapillaries and lymph system thereby allowing tumor cells to invade intothe target tissues (Dano, et al. Adv. Cancer. Res., 44:139 (1985)).Thus, it is within the scope of the present invention to utilize as alinker a peptide sequence that is cleaved by urokinase.

The invention also provides the use of peptide sequences that aresensitive to cleavage by tryptases. Human mast cells express at leastfour distinct tryptases, designated αβI, βII, and βIII. These enzymesare not controlled by blood plasma proteinase inhibitors and only cleavea few physiological substrates in vitro. The tryptase family of serineproteases has been implicated in a variety of allergic and inflammatorydiseases involving mast cells because of elevated tryptase levels foundin biological fluids from patients with these disorders. However, theexact role of tryptase in the pathophysiology of disease remains to bedelineated. The scope of biological functions and correspondingphysiological consequences of tryptase are substantially defined bytheir substrate specificity.

Tryptase is a potent activator of pro-urokinase plasminogen activator(uPA), the zymogen form of a protease associated with tumor metastasisand invasion. Activation of the plasminogen cascade, resulting in thedestruction of extracellular matrix for cellular extravasation andmigration, may be a function of tryptase activation of pro-urokinaseplasminogen activator at the P4-P1 sequence of Pro-Arg-Phe-Lys (SEQ. IDNO: 98) (Stack, et al., Journal of Biological Chemistry 269 (13):9416-9419 (1994)). Vasoactive intestinal peptide, a neuropeptide that isimplicated in the regulation of vascular permeability, is also cleavedby tryptase, primarily at the Thr-Arg-Leu-Arg (SEQ. ID NO: 99) sequence(Tam, et al., Am. J. Respir. Cell Mol. Biol. 3: 27-32 (1990)). TheG-protein coupled receptor PAR-2 can be cleaved and activated bytryptase at the Ser-Lys-Gly-Arg (SEQ. ID NO: 100) sequence to drivefibroblast proliferation, whereas the thrombin activated receptor PAR-1is inactivated by tryptase at the Pro-Asn-Asp-Lys (SEQ. ID NO: 101)sequence (Molino et al., Journal of Biological Chemistry 272(7):4043-4049 (1997)). Taken together, this evidence suggests a central rolefor tryptase in tissue remodeling as a consequence of disease. This isconsistent with the profound changes observed in several mastcell-mediated disorders. One hallmark of chronic asthma and otherlong-term respiratory diseases is fibrosis and thickening of theunderlying tissues that could be the result of tryptase activation ofits physiological targets. Similarly, a series of reports have shownangiogenesis to be associated with mast cell density, tryptase activityand poor prognosis in a variety of cancers (Coussens et al., Genes andDevelopment 13(11): 1382-97 (1999)); Takanami et al., Cancer 88(12):2686-92 (2000); Toth-Jakatics et al., Human Pathology 31(8): 955-960(2000); Ribatti et al., International Journal of Cancer 85(2): 171-5(2000)).

Methods are known in the art for evaluating whether a particularprotease cleaves a selected peptide sequence. For example, the use of7-amino-4-methyl coumarin (AMC) fluorogenic peptide substrates is awell-established method for the determination of protease specificity(Zimmerman, M., et al., (1977) Analytical Biochemistry 78:47-51).Specific cleavage of the anilide bond liberates the fluorogenic AMCleaving group allowing for the simple determination of cleavage ratesfor individual substrates. More recently, arrays (Lee, D., et al.,(1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) andpositional-scanning libraries (Rano, T. A., et al., (1997) Chemistry andBiology 4:149-55) of AMC peptide substrate libraries have been employedto rapidly profile the N-terminal specificity of proteases by sampling awide range of substrates in a single experiment. Thus, one of skill inthe art may readily evaluate an array of peptide sequences to determinetheir utility in the present invention without resort to undueexperimentation.

The antibody-partner conjugate of the current invention may optionallycontain two or more linkers. These linkers may be the same or different.For example, a peptidyl linker may be used to connect the drug to theligand and a second peptidyl linker may attach a diagnostic agent to thecomplex. Other uses for additional linkers include linking analyticalagents, biomolecules, targeting agents, and detectable labels to theantibody-partner complex.

Also within the scope of the present invention are compounds of theinvention that are poly- or multi-valent species, including, forexample, species such as dimers, trimers, tetramers and higher homologsof the compounds of the invention or reactive analogues thereof. Thepoly- and multi-valent species can be assembled from a single species ormore than one species of the invention. For example, a dimeric constructcan be “homo-dimeric” or “heterodimeric.” Moreover, poly- andmulti-valent constructs in which a compound of the invention or areactive analogue thereof, is attached to an oligomeric or polymericframework (e.g., polylysine, dextran, hydroxyethyl starch and the like)are within the scope of the present invention. The framework ispreferably polyfunctional (i.e. having an array of reactive sites forattaching compounds of the invention). Moreover, the framework can bederivatized with a single species of the invention or more than onespecies of the invention.

Moreover, the present invention includes compounds that arefunctionalized to afford compounds having water-solubility that isenhanced relative to analogous compounds that are not similarlyfunctionalized. Thus, any of the substituents set forth herein can bereplaced with analogous radicals that have enhanced water solubility.For example, it is within the scope of the invention to, for example,replace a hydroxyl group with a diol, or an amine with a quaternaryamine, hydroxy amine or similar more water-soluble moiety. In apreferred embodiment, additional water solubility is imparted bysubstitution at a site not essential for the activity towards the ionchannel of the compounds set forth herein with a moiety that enhancesthe water solubility of the parent compounds. Methods of enhancing thewater-solubility of organic compounds are known in the art. Such methodsinclude, but are not limited to, functionalizing an organic nucleus witha permanently charged moiety, e.g., quaternary ammonium, or a group thatis charged at a physiologically relevant pH, e.g. carboxylic acid,amine. Other methods include, appending to the organic nucleus hydroxyl-or amine-containing groups, e.g. alcohols, polyols, polyethers, and thelike. Representative examples include, but are not limited to,polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art. See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991.

Hydrazine Linkers (H)

In a second embodiment, the conjugate of the invention comprises ahydrazine self-immolative linker, wherein the conjugate has thestructure:

X⁴-(L⁴)_(p)-H-(L¹)_(m)-D

wherein D, L¹, L⁴, and X⁴ are as defined above and described furtherherein, and H is a linker comprising the structure:

wherein n₁ is an integer from 1-10; n₂ is 0, 1, or 2; each R²⁴ is amember independently selected from the group consisting of H,substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, andunsubstituted heteroalkyl; and I is either a bond (i.e., the bondbetween the carbon of the backbone and the adjacent nitrogen) or:

wherein n₃ is 0 or 1, with the proviso that when n₃ is 0, n₂ is not 0;and n₄ is 1, 2, or 3, wherein when I is a bond, n₁ is 3 and n₂ is 1, Dcan not be

where R is Me or CH₂—CH₂—NMe₂.

In one embodiment, the substitution on the phenyl ring is a parasubstitution. In preferred embodiments, n₁ is 2, 3, or 4 or n₁ is 3. Inpreferred embodiments, n₂ is 1. In preferred embodiments, I is a bond(i.e., the bond between the carbon of the backbone and the adjacentnitrogen). In one aspect, the hydrazine linker, H, can form a 6-memberedself immolative linker upon cleavage, for example, when n₃ is 0 and n4is 2. In another aspect, the hydrazine linker, H, can form two5-membered self immolative linkers upon cleavage. In yet other aspects,H forms a 5-membered self immolative linker, H forms a 7-membered selfimmolative linker, or H forms a 5-membered self immolative linker and a6-membered self immolative linker, upon cleavage. The rate of cleavageis affected by the size of the ring formed upon cleavage. Thus,depending upon the rate of cleavage desired, an appropriate size ring tobe formed upon cleavage can be selected.

Five Membered Hydrazine Linkers

In one embodiment, the hydrazine linker comprises a 5-membered hydrazinelinker, wherein H comprises the structure:

In a preferred embodiment, n₁ is 2, 3, or 4. In another preferredembodiment, n₁ is 3.

In the above structure, each R²⁴ is a member independently selected fromthe group consisting of H, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, and unsubstituted heteroalkyl. In oneembodiment, each R²⁴ is independently H or a C₁-C₆ alkyl. In anotherembodiment, each R²⁴ is independently H or a C₁-C₃ alkyl, morepreferably H or CH₃. In another embodiment, at least one R²⁴ is a methylgroup. In another embodiment, each R₂₄ is H. Each R²⁴ is selected totailor the compounds steric effects and for altering solubility.

The 5-membered hydrazine linkers can undergo one or more cyclizationreactions that separate the drug from the linker, and can be described,for example, by:

An exemplary synthetic route for preparing a five membered linker of theinvention is:

The Cbz-protected DMDA b is reacted with 2,2-Dimethyl-malonic acid a insolution with thionyl chloride to form a Cbz-DMDA-2,2-dimethylmalonicacid c. Compound c is reacted with Boc-N-methyl hydrazine d in thepresence of EDC to form DMDA-2,2-dimetylmalonic-Boc-N-methylhydrazine e.

Six Membered Hydrazine Linkers

In another embodiment, the hydrazine linker comprises a 6-memberedhydrazine linker, wherein H comprises the structure:

In a preferred embodiment, n₁ is 3. In the above structure, each R²⁴ isa member independently selected from the group consisting of H,substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, andunsubstituted heteroalkyl. In one embodiment, each R²⁴ is independentlyH or a C₁-C₆ alkyl. In another embodiment, each R²⁴ is independently Hor a C₁-C₃ alkyl, more preferably H or CH₃. In another embodiment, atleast one R²⁴ is a methyl group. In another embodiment, each R₂₄ is H.Each R²⁴ is selected to tailor the compounds steric effects and foraltering solubility. In a preferred embodiment, H comprises thestructure:

In one embodiment, H comprises a geminal dimethyl substitution. In oneembodiment of the above structure, each R²⁴ is independently an H or asubstituted or unsubstituted alkyl.

The 6-membered hydrazine linkers will undergo a cyclization reactionthat separates the drug from the linker, and can be described as:

An exemplary synthetic route for preparing a six membered linker of theinvention is:

The Cbz-protected dimethyl alanine a in solution with dichlormethane,was reacted with HOAt, and CPI to form a Cbz-protected dimethylalaninehydrazine b. The hydrazine b is deprotected by the action of methanol,forming compound c.

Other Hydrazine Linkers

It is contemplated that the invention comprises a linker having sevenmembers. This linker would likely not cyclize as quickly as the five orsix membered linkers, but this may be preferred for someantibody-partner conjugates. Similarly, the hydrazine linker maycomprise two six membered rings or a hydrazine linker having one six andone five membered cyclization products. A five and seven membered linkeras well as a six and seven membered linker are also contemplated.

Another hydrazine structure, H, has the formula:

where q is 0, 1,2, 3, 4, 5, or 6; and

each R²⁴ is a member independently selected from the group consisting ofH, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, andunsubstituted heteroalkyl. This hydrazine structure can also form five-,six-, or seven-membered rings and additional components can be added toform multiple rings.

Disulfide Linkers (J)

In yet another embodiment, the linker comprises an enzymaticallycleavable disulfide group. In one embodiment, the invention provides acytotoxic antibody-partner compound having a structure according toFormula (d):

wherein D, L¹, L⁴, and X⁴ are as defined above and described furtherherein, and J is a disulfide linker comprising a group having thestructure:

wherein each R²⁴ is a member independently selected from the groupconsisting of H, substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, and unsubstituted heteroalkyl; each K is a memberindependently selected from the group consisting of substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, NO₂, NR²¹R²², NR²¹COR²², OCONR²¹R²², OCOR²¹,and OR²¹ wherein R²¹ and R²² are independently selected from the groupconsisting of H, substituted alkyl, unsubstituted alkyl, substitutedheteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedheterocycloalkyl and unsubstituted heterocycloalkyl; i is an integer of0, 1, 2, 3, or 4; and d is an integer of 0, 1, 2, 3, 4, 5, or 6.

The aromatic ring of the disulfides linker may be substituted with oneor more “K” groups. A “K” group is a substituent on the aromatic ringthat replaces a hydrogen otherwise attached to one of the fournon-substituted carbons that are part of the ring structure. The “K”group may be a single atom, such as a halogen, or may be a multi-atomgroup, such as alkyl, heteroalkyl, amino, nitro, hydroxy, alkoxy,haloalkyl, and cyano. Exemplary K substituents independently include,but are not limited to, F, Cl, Br, I, NO₂, OH, OCH₃, NHCOCH₃, N(CH₃)₂,NHCOCF₃ and methyl. For “K_(i)”, i is an integer of 0, 1, 2, 3, or 4. Ina specific embodiment, i is 0.

In a preferred embodiment, the linker comprises an enzymaticallycleavable disulfide group of the following formula:

In this embodiment, the identities of L⁴, X⁴, p, and R²⁴ are asdescribed above, and d is 0, 1, 2, 3, 4, 5, or 6. In a particularembodiment, d is 1 or 2.

A more specific disulfide linker is shown in the formula below:

A specific example of this embodiment is as follows:

Preferably, d is 1 or 2.

Another disulfide linker is shown in the formula below:

A specific example of this embodiment is as follows:

Preferably, d is 1 or 2.

In various embodiments, the disulfides are ortho to the amine. Inanother specific embodiment, a is 0. In preferred embodiments, R²⁴ isindependently selected from H and CH₃.

An exemplary synthetic route for preparing a disulfide linker of theinvention is as follows:

A solution of 3-mercaptopropionic acid a is reacted with aldrithiol-2 toform 3-methyl benzothiazolium iodide b. 3-methylbenzothiazolium iodide cis reacted with sodium hydroxide to form compound d. A solution ofcompound d with methanol is further reacted with compound b to formcompound e. Compound e deprotected by the action of acetyl chloride andmethanol forming compound f.

For further discussion of types of cytotoxins, linkers and other methodsfor conjugating therapeutic agents to antibodies, see also PCTPublication WO 2007/059404 to Gangwar et al. and entitled “CytotoxicCompounds And Conjugates,” Saito, G. et al. (2003) Adv. Drug Deliv. Rev.55:199-215; Trail, P. A. et al. (2003) Cancer Immunol. Immunother.52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002)Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr.Opin. Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.(2001) Adv. Drug Deliv. Rev. 53:247-264, each of which are herebyincorporated by reference in their entirety.

Partner Molecules

In one aspect, the present invention features an antibody conjugated toa partner molecule, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Such conjugates are also referred toherein as “immunoconjugates.” Immunoconjugates that include one or morecytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells.

Examples of partner molecules of the present invention include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Examples of partner molecules also include, forexample, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

Other preferred examples of partner molecules that can be conjugated toan antibody of the invention include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg®;American Home Products).

Preferred examples of partner molecule are CC-1065 and the duocarmycins.CC-1065 was first isolated from Streptomyces zelensis in 1981 by theUpjohn Company (Hanka et al., J. Antibiot. 31: 1211 (1978); Martin etal., J. Antibiot. 33: 902 (1980); Martin et al., J. Antibiot. 34: 1119(1981)) and was found to have potent antitumor and antimicrobialactivity both in vitro and in experimental animals (Li et al., CancerRes. 42: 999 (1982)). CC-1065 binds to double-stranded B-DNA within theminor groove (Swenson et al., Cancer Res. 42: 2821 (1982)) with thesequence preference of 5′-d(A/GNTTA)-3′ and 5′-d(AAAAA)-3′ and alkylatesthe N3 position of the 3′-adenine by its CPI left-hand unit present inthe molecule (Hurley et al., Science 226: 843 (1984)). Despite itspotent and broad antitumor activity, CC-1065 cannot be used in humansbecause it causes delayed death in experimental animals.

Many analogues and derivatives of CC-1065 and the duocarmycins are knownin the art. The research into the structure, synthesis and properties ofmany of the compounds has been reviewed. See, for example, Boger et al.,Angew. Chem. Int. Ed. Engl. 35: 1438 (1996); and Boger et al., Chem.Rev. 97: 787 (1997).

A group at Kyowa Hakko Kogya Co., Ltd. has prepared a number of CC-1065derivatives. See, for example, U.S. Pat. Nos. 5,101,038; 5,641,780;5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; and5,084,468; and published PCT application, WO 96/10405 and publishedEuropean application 0 537 575 A1.

The Upjohn Company (Pharmacia Upjohn) has also been active in preparingderivatives of CC-1065. See, for example, U.S. Pat. Nos. 5,739,350;4,978,757, 5,332, 837 and 4,912,227.

A particularly preferred aspect of the current invention provides acytotoxic compound having a structure according to the following formula(e):

in which ring system A is a member selected from substituted orunsubstituted aryl substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups. Exemplary ringsystems include phenyl and pyrrole.

The symbols E and G are independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, aheteroatom, a single bond or E and G are optionally joined to form aring system selected from substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl and substituted or unsubstitutedheterocycloalkyl.

The symbol X represents a member selected from O, S and NR²³. R²³ is amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl.

The symbol R³ represents a member selected from (═O), SR¹¹, NHR¹¹ andOR¹¹, in which R¹¹ is H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, monophosphates, diphosphates,triphosphates, sulfonates, acyl, C(O)R¹²R¹³, C(O)OR¹², C(O)NR¹²R¹³,P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² or SiR¹²R¹³R¹⁴. The symbols R¹², R¹³,and R¹⁴ independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl and substituted orunsubstituted aryl, where R¹² and R¹³ together with the nitrogen orcarbon atom to which they are attached are optionally joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms. One or moreof R¹², R¹³, or R¹⁴ can include a cleavable group within its structure.

R⁴, R^(4′), R⁵ and R^(5′) are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶,OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, and O(CH₂)_(n)N(CH₃)₂, wheren is an integer from 1 to 20, or any adjacent pair of R⁴, R^(4′), R⁵ andR^(5′), together with the carbon atoms to which they are attached, arejoined to form a substituted or unsubstituted cycloalkyl orheterocycloalkyl ring system having from 4 to 6 members. R¹⁵ and R¹⁶independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl and substituted or unsubstitutedpeptidyl, where R¹⁵ and R¹⁶ together with the nitrogen atom to whichthey are attached are optionally joined to form a substituted orunsubstituted heterocycloalkyl ring system having from 4 to 6 members,optionally containing two or more heteroatoms. One exemplary structureis aniline.

R⁴, R^(4′), R⁵, R^(5′), R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ optionally containone or more cleavable groups within their structure, such as a cleavablelinker or cleavable substrate. Exemplary cleavable groups include, butare not limited to peptides, amino acids, hydrazines, disulfides, andcephalosporin derivatives.

In some embodiments, at least one of R⁴, R^(4′), R⁵, R^(5′), R¹¹, R¹²,R¹³, R¹⁵ and R¹⁶ is used to join the drug to a linker or enzymecleavable substrate of the present invention, as described herein, forexample to L¹, if present or to F, H, J, or X², or J.

In a still further exemplary embodiment, at least one of R⁴, R^(4′), R⁵,R^(5′), R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ bears a reactive group appropriatefor conjugating the compound. In a further exemplary embodiment, R⁴,R^(4′), R⁵, R^(5′), R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ are independentlyselected from H, substituted alkyl and substituted heteroalkyl and havea reactive functional group at the free terminus of the alkyl orheteroalkyl moiety. One or more of R⁴, R^(4′), R⁵, R^(5′), R¹¹, R¹²,R¹³, R¹⁵ and R¹⁶ may be conjugated to another species, e.g, targetingagent, detectable label, solid support, etc.

R⁶ is a single bond which is either present or absent. When R⁶ ispresent, R⁶ and R⁷ are joined to form a cyclopropyl ring. R⁷ is CH₂—X¹or —CH₂—. When R⁷ is —CH₂— it is a component of the cyclopropane ring.The symbol X¹ represents a leaving group such as a halogen, for exampleCl, Br or F. The combinations of R⁶ and R⁷ are interpreted in a mannerthat does not violate the principles of chemical valence.

X¹ may be any leaving group. Useful leaving groups include, but are notlimited to, halogens, azides, sulfonic esters (e.g., alkylsulfonyl,arylsulfonyl), oxonium ions, alkyl perchlorates, ammonioalkanesulfonateesters, alkylfluorosulfonates and fluorinated compounds (e.g.,triflates, nonaflates, tresylates) and the like. Particular halogensuseful as leaving groups are F, Cl and Br. The choice of these and otherleaving groups appropriate for a particular set of reaction conditionsis within the abilities of those of skill in the art (see, for example,March J, Advanced Organic Chemistry, 2nd Edition, John Wiley and Sons,1992; Sandler S R, Karo W, Organic Functional Group Preparations, 2ndEdition, Academic Press, Inc., 1983; and Wade L G, Compendium of OrganicSynthetic Methods, John Wiley and Sons, 1980).

The curved line within the six-membered ring indicates that the ring mayhave one or more degrees of unsaturation, and it may be aromatic. Thus,ring structures such as those set forth below, and related structures,are within the scope of Formula (f):

In some embodiments, at least one of R⁴, R^(4′), R⁵, and R^(5′) linkssaid drug to L¹, if present, or to F, H, J, or X², and includes

where v is an integer from 1 to 6; and each R²⁷, R^(27′), R²⁸, andR^(28′) is independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted heterocycloalkyl. In some embodiments, R²⁷,R^(27′), R²⁸, and R^(28′) are all H. In some embodiments, v is aninteger from 1 to 3 (preferably, 1). This unit can be used to separatearyl substituents from the drug and thereby resist or avoid generatingcompounds that are substrates for multi-drug resistance.

In one embodiment, R¹¹ includes a moiety, X⁵, that does not self-cyclizeand links the drug to L, if present, or to F, H, J, or X². The moiety,X⁵, is preferably cleavable using an enzyme and, when cleaved, providesthe active drug. As an example, R¹¹ can have the following structure(with the right side coupling to the remainder of the drug):

In an exemplary embodiment, ring system A of formula (e) is asubstituted or unsubstituted phenyl ring. Ring system A may besubstituted with one or more aryl group substituents as set forth in thedefinitions section herein. In some embodiments, the phenyl ring issubstituted with a CN or methoxy moiety.

In some embodiments, at least one of R⁴, R^(4′), R⁵, and R^(5′) linkssaid drug to L¹, if present, or to F, H, J, or X², and R³ is selectedfrom SR¹¹, NHR¹¹ and OR¹¹. R¹¹ is selected from —SO(OH)₂, —PO(OH)₂,-AA_(n), —Si(CH₃)₂C(CH₃)₃, —C(O)OPhNH(AA)_(m),

or any other sugar or combination of sugars,

and pharmaceutically acceptable salts thereof, where n is any integer inthe range of 1 to 10, m is any integer in the range of 1 to 4, p is anyinteger in the range of 1 to 6, and AA is any natural or non-naturalamino acid. In some embodiments, AA_(n) or AA_(m) is selected from thesame amino acid sequences described above for the peptide linkers (F)and optionally is the same as the amino acid sequence used in the linkerportion of R⁴, R^(4′), R⁵, or R^(5′). In at least some embodiments, R³is cleavable in vivo to provide an active drug compound. In at leastsome embodiments, R³ increases in vivo solubility of the compound. Insome embodiments, the rate of decrease of the concentration of theactive drug in the blood is substantially faster than the rate ofcleavage of R³ to provide the active drug. This may be particularlyuseful where the toxicity of the active drug is substantially higherthan that of the prodrug form. In other embodiments, the rate ofcleavage of R³ to provide the active drug is faster than the rate ofdecrease of concentration of the active drug in the blood.

In another exemplary embodiment, the invention provides a compoundhaving a structure according to Formula (g):

In this embodiment, the identities of the substituents R³, R⁴, R^(4′),R⁵, R^(5′), R⁶, R⁷ and X are substantially as described above forFormula (a), as well as preferences for particular embodiments. Thesymbol Z is a member independently selected from O, S and NR²³. Thesymbol R²³ represents a member selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl.Each R²³ is independently selected. The symbol R¹ represents H,substituted or unsubstituted lower alkyl, or C(O)R⁸ or CO₂R⁸. R⁸ is amember selected from substituted alkyl, unsubstituted alkyl, NR⁹R¹⁰,NR⁹NHR¹⁰ and OR⁹. R⁹ and R¹⁰ are independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. R² is H, or substituted or unsubstituted lower alkyl. It isgenerally preferred that when R² is substituted alkyl, it is other thana perfluoroalkyl, e.g., CF₃. In one embodiment, R² is a substitutedalkyl wherein the substitution is not a halogen. In another embodiment,R² is an unsubstituted alkyl.

In some embodiments R¹ is an ester moiety, such as CO₂CH₃. In someembodiments, R² is a lower alkyl group, which may be substituted orunsubstituted. A presently preferred lower alkyl group is CH₃. In somepreferred embodiments, R¹ is CO₂CH₃ and R² is CH₃.

In some embodiments, R⁴, R^(4′), R⁵, and R^(5′) are membersindependently selected from H, halogen, NH₂, OMe, O(CH₂)₂N(R²⁹)₂ andNO₂. Each R²⁹ is independently H or lower alkyl (e.g., methyl).

In some embodiments, the drug is selected such that the leaving group X¹is a member selected from the group consisting of halogen,alkylsulfonyl, arylsulfonyl, and azide. In some embodiments, X¹ is F,Cl, or Br.

In some embodiments, Z is O or NH. In some embodiments, X is O.

In yet another exemplary embodiment, the invention provides compoundshaving a structure according to Formula (h) or (i):

Another preferred structure of the duocarmycin analog of Formula (e) isa structure in which the ring system A is an unsubstituted orsubstituted phenyl ring. The preferred substituents on the drug moleculedescribed hereinabove for the structure of Formula 7 when the ringsystem A is a pyrrole are also preferred substituents when the ringsystem A is an unsubstituted or substituted phenyl ring.

For example, in a preferred embodiment, the drug (D) comprises astructure (j):

In this structure, R³, R⁶, R⁷, X are as described above for Formula (e).Furthermore, Z is a member selected from O, S and NR²³, wherein R²³ is amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl;

R¹ is H, substituted or unsubstituted lower alkyl, C(O)R⁸, or CO₂R⁸,wherein R⁸ is a member selected from NR⁹R¹⁰ and OR⁹, in which R⁹ and R¹⁰are members independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl;

R^(1′) is H, substituted or unsubstituted lower alkyl, or C(O)R⁸,wherein R⁸ is a member selected from NR⁹R¹⁰ and OR⁹, in which R⁹ and R¹⁰are members independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl;

R² is H, or substituted or unsubstituted lower alkyl or unsubstitutedheteroalkyl or cyano or alkoxy; and R^(2′) is H, or substituted orunsubstituted lower alkyl or unsubstituted heteroalkyl.

At least one of R⁴, R^(4′), R⁵, R^(5′), R¹¹, R¹², R¹³, R¹⁵ or R¹⁶ linksthe drug to L¹, if present, or to F, H, J, or X².

Another embodiment of the drug (D) comprises a structure (k) where R⁴and R^(4′) have been joined to from a heterocycloalkyl:

In this structure, R³, R⁵, R^(5′), R⁶, R⁷, X are as described above forFormula (e). Furthermore, Z is a member selected from O, S and NR²³,wherein R²³ is a member selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, and acyl;

R³² is selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶,NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, andO(CH₂)_(n)N(CH₃)₂, where n is an integer from 1 to 20. R¹⁵ and R¹⁶independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl and substituted or unsubstitutedpeptidyl, where R¹⁵ and R¹⁶ together with the nitrogen atom to whichthey are attached are optionally joined to form a substituted orunsubstituted heterocycloalkyl ring system having from 4 to 6 members,optionally containing two or more heteroatoms. R³² optionally containsone or more cleavable groups within its structure, such as a cleavablelinker or cleavable substrate. Exemplary cleavable groups include, butare not limited to, peptides, amino acids, hydrazines, disulfides, andcephalosporin derivatives. Moreover, any selection of substituentsdescribed herein for R⁴, R^(4′), R⁵, R^(5′), R¹⁵, and R¹⁶ is alsoapplicable to R³².

At least one of R⁵, R^(5′), R¹¹, R¹², R¹³, R¹⁵, R¹⁶, or R³² links thedrug to L¹, if present, or to F, H, J, or X². In at least someembodiments, R³² links the drug to L¹, if present, or to F, H, J, or X².

One preferred embodiment of this compound is:

R¹ is H, substituted or unsubstituted lower alkyl, C(O)R⁸, or CO₂R⁸,wherein R⁸ is a member selected from NR⁹R¹⁰ and OR⁹, in which R⁹ and R¹⁰are members independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl;

R^(1′) is H, substituted or unsubstituted lower alkyl, or C(O)R⁸,wherein R⁸ is a member selected from NR⁹R¹⁰ and OR⁹, in which R⁹ and R¹⁰are members independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl;

R² is H, or substituted or unsubstituted lower alkyl or unsubstitutedheteroalkyl or cyano or alkoxy; and R^(2′) is H, or substituted orunsubstituted lower alkyl or unsubstituted heteroalkyl.

A further embodiment has the formula:

In this structure, A, R⁶, R⁷, X, R⁴, R^(4′), R⁵, and R^(3′) are asdescribed above for Formula (e). Furthermore, Z is a member selectedfrom O, S and NR²³, where R²³ is a member selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl, andacyl;

R³³ is selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶,NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, andO(CH₂)_(n)N(CH₃)₂, where n is an integer from 1 to 20. R¹⁵ and R¹⁶independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl and substituted or unsubstitutedpeptidyl, where R¹⁵ and R¹⁶ together with the nitrogen atom to whichthey are attached are optionally joined to form a substituted orunsubstituted heterocycloalkyl ring system having from 4 to 6 members,optionally containing two or more heteroatoms. R³³ links the drug to L,if present, or to F, H, J, or X².

Preferably, A is substituted or unsubstituted phenyl or substituted orunsubstituted pyrrole. Moreover, any selection of substituents describedherein for R¹¹ is also applicable to R³³.

Ligands

X⁴ represents a ligand selected from the group consisting of protectedreactive functional groups, unprotected reactive functional groups,detectable labels, and targeting agents. Preferred ligands are targetingagents, such as antibodies and fragments thereof.

In some embodiments, the group X⁴ can be described as a member selectedfrom R²⁹, COOR²⁹, C(O)NR²⁹, and C(O)NNR²⁹ wherein R²⁹ is a memberselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl and substituted or unsubstituted heteroaryl.In yet another exemplary embodiment, R²⁹ is a thiol reactive member. Ina further exemplary embodiment, R²⁹ is a thiol reactive member selectedfrom haloacetyl and alkyl halide derivatives, maleimides, aziridines,and acryloyl derivatives. The above thiol reactive members can act asreactive protective groups that can be reacted with, for example, a sidechain of an amino acid of a targeting agent, such as an antibody, tothereby link the targeting agent to the linker-drug moiety.

Detectable Labels

The particular label or detectable group used in conjunction with thecompounds and methods of the invention is generally not a criticalaspect of the invention, as long as it does not significantly interferewith the activity or utility of the compound of the invention. Thedetectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to a compound of theinvention according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

When the compound of the invention is conjugated to a detectable label,the label is preferably a member selected from the group consisting ofradioactive isotopes, fluorescent agents, fluorescent agent precursors,chromophores, enzymes and combinations thereof. Methods for conjugatingvarious groups to antibodies are well known in the art. For example, adetectable label that is frequently conjugated to an antibody is anenzyme, such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, and glucose oxidase.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to a component ofthe conjugate. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound.

Components of the conjugates of the invention can also be conjugateddirectly to signal generating compounds, e.g., by conjugation with anenzyme or fluorophore. Enzymes of interest as labels will primarily behydrolases, particularly phosphatases, esterases and glycosidases, oroxidotases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Fluorescent labels are presently preferred as they have the advantage ofrequiring few precautions in handling, and being amenable tohigh-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Preferred labels are typically characterized byone or more of the following: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling. Many fluorescent labels are commercially available from theSIGMA chemical company (Saint Louis, Mo.), Molecular Probes (Eugene,Oreg.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), and Applied Biosystems (Foster City, Calif.), as well asmany other commercial sources known to one of skill. Furthermore, thoseof skill in the art will recognize how to select an appropriatefluorophore for a particular application and, if it not readilyavailable commercially, will be able to synthesize the necessaryfluorophore de novo or synthetically modify commercially availablefluorescent compounds to arrive at the desired fluorescent label.

In addition to small molecule fluorophores, naturally occurringfluorescent proteins and engineered analogues of such proteins areuseful in the present invention. Such proteins include, for example,green fluorescent proteins of cnidarians (Ward et al., Photochem.Photobiol. 35:803-808 (1982); Levine et al., Comp. Biochem. Physiol.,72B:77-85 (1982)), yellow fluorescent protein from Vibrio fischeristrain (Baldwin et al., Biochemistry 29:5509-15 (1990)),Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp. (Morriset al., Plant Molecular Biology 24:673:77 (1994)), phycobiliproteinsfrom marine cyanobacteria, such as Synechococcus, e.g., phycoerythrinand phycocyanin (Wilbanks et al., J. Biol. Chem. 268:1226-35 (1993)),and the like.

Generally, prior to forming the linkage between the cytoCytotoxin Andthe targeting (or other) agent, and optionally, the spacer group, atleast one of the chemical functionalities will be activated. One skilledin the art will appreciate that a variety of chemical functionalities,including hydroxy, amino, and carboxy groups, can be activated using avariety of standard methods and conditions. For example, a hydroxylgroup of the cytotoxin or targeting agent can be activated throughtreatment with phosgene to form the corresponding chloroformate, orp-nitrophenylchloroformate to form the corresponding carbonate.

In an exemplary embodiment, the invention makes use of a targeting agentthat includes a carboxyl functionality. Carboxyl groups may be activatedby, for example, conversion to the corresponding acyl halide or activeester. This reaction may be performed under a variety of conditions asillustrated in March, supra pp. 388-89. In an exemplary embodiment, theacyl halide is prepared through the reaction of the carboxyl-containinggroup with oxalyl chloride. The activated agent is reacted with acytotoxin or cytotoxin-linker arm combination to form a conjugate of theinvention. Those of skill in the art will appreciate that the use ofcarboxyl-containing targeting agents is merely illustrative, and thatagents having many other functional groups can be conjugated to thelinkers of the invention.

Reactive Functional Groups

For clarity of illustration the succeeding discussion focuses on theconjugation of a cytotoxin of the invention to a targeting agent. Thefocus exemplifies one embodiment of the invention from which, others arereadily inferred by one of skill in the art. No limitation of theinvention is implied, by focusing the discussion on a single embodiment.

Exemplary compounds of the invention bear a reactive functional group,which is generally located on a substituted or unsubstituted alkyl orheteroalkyl chain, allowing their facile attachment to another species.A convenient location for the reactive group is the terminal position ofthe chain.

Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. The reactive functional group may be protectedor unprotected, and the protected nature of the group may be changed bymethods known in the art of organic synthesis. Preferred classes ofreactions available with reactive cytoCytotoxin Analogues are thosewhich proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, SanDiego, 1996; and Feeney et al., Modification of Proteins; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

Exemplary reaction types include the reaction of carboxyl groups andvarious derivatives thereof including, but not limited to,N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides,acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl,alkynyl and aromatic esters. Hydroxyl groups can be converted to esters,ethers, aldehydes, etc. Haloalkyl groups are converted to new species byreaction with, for example, an amine, a carboxylate anion, thiol anion,carbanion, or an alkoxide ion. Dienophile (e.g., maleimide) groupsparticipate in Diels-Alder. Aldehyde or ketone groups can be convertedto imines, hydrazones, semicarbazones or oximes, or via such mechanismsas Grignard addition or alkyllithium addition. Sulfonyl halides reactreadily with amines, for example, to form sulfonamides. Amine orsulfhydryl groups are, for example, acylated, alkylated or oxidized.Alkenes, can be converted to an array of new species usingcycloadditions, acylation, Michael addition, etc. Epoxides react readilywith amines and hydroxyl compounds.

One skilled in the art will readily appreciate that many of theselinkages may be produced in a variety of ways and using a variety ofconditions. For the preparation of esters, see, e.g., March supra at1157; for thioesters, see, March, supra at 362-363, 491, 720-722, 829,941, and 1172; for carbonates, see, March, supra at 346-347; forcarbamates, see, March, supra at 1156-57; for amides, see, March supraat 1152; for ureas and thioureas, see, March supra at 1174; for acetalsand ketals, see, Greene et al. supra 178-210 and March supra at 1146;for acyloxyalkyl derivatives, see, Prodrugs: Topical and Ocular DrugDelivery, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; forenol esters, see, March supra at 1160; for N-sulfonylimidates, see,Bundgaard et al., J. Med. Chem., 31:2066 (1988); for anhydrides, see,March supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see,March supra at 379; for N-Mannich bases, see, March supra at 800-02, and828; for hydroxymethyl ketone esters, see, Petracek et al. Annals NYAcad. Sci., 507:353-54 (1987); for disulfides, see, March supra at 1160;and for phosphonate esters and phosphonamidates.

The reactive functional groups can be unprotected and chosen such thatthey do not participate in, or interfere with, the reactions.Alternatively, a reactive functional group can be protected fromparticipating in the reaction by the presence of a protecting group.Those of skill in the art will understand how to protect a particularfunctional group from interfering with a chosen set of reactionconditions. For examples of useful protecting groups, See Greene et al.,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

Typically, the targeting agent is linked covalently to a cytotoxin usingstandard chemical techniques through their respective chemicalfunctionalities. Optionally, the linker or agent is coupled to the agentthrough one or more spacer groups. The spacer groups can be equivalentor different when used in combination.

Generally, prior to forming the linkage between the cytoCytotoxin Andthe reactive functional group, and optionally, the spacer group, atleast one of the chemical functionalities will be activated. One skilledin the art will appreciate that a variety of chemical functionalities,including hydroxy, amino, and carboxy groups, can be activated using avariety of standard methods and conditions. In an exemplary embodiment,the invention comprises a carboxyl functionality as a reactivefunctional group. Carboxyl groups may be activated as describedhereinabove.

Cleavable Substrate

The cleavable substrates of the current invention are depicted as “X²”.Preferably, the cleavable substrate is a cleavable enzyme substrate thatcan be cleaved by an enzyme. Preferably, the enzyme is preferentiallyassociated, directly or indirectly, with the tumor or other target cellsto be treated. The enzyme may be generated by the tumor or other targetcells to be treated. For example, the cleavable substrate can be apeptide that is preferentially cleavable by an enzyme found around or ina tumor or other target cell. Additionally or alternatively, the enzymecan be attached to a targeting agent that binds specifically to tumorcells, such as an antibody specific for a tumor antigen.

As examples of enzyme cleavable substrates suitable for coupling to thedrugs described above, PCT Patent Applications Publication Nos. WO00/33888, WO 01/95943, WO 01/95945, WO 02/00263, and WO 02/100353, allof which are incorporated herein by reference, disclose attachment of acleavable peptide to a drug. The peptide is cleavable by an enzyme, suchas a trouase (such as thimet oligopeptidase), CD10 (neprilysin), amatrix metalloprotease (such as MMP2 or MMP9), a type II transmembraneserine protease (such as Hepsin, testisin, TMPRSS4, ormatriptase/MT-SP1), or a cathepsin, associated with a tumor. In thisembodiment, a prodrug includes the drug as described above, a peptide, astabilizing group, and optionally a linking group between the drug andthe peptide. The stabilizing group is attached to the end of the peptideto protect the prodrug from degradation before arriving at the tumor orother target cell. Examples of suitable stabilizing groups includenon-amino acids, such as succinic acid, diglycolic acid, maleic acid,polyethylene glycol, pyroglutamic acid, acetic acid, naphthylcarboxylicacid, terephthalic acid, and glutaric acid derivatives; as well asnon-genetically-coded amino acids or aspartic acid or glutamic acidattached to the N-terminus of the peptide at the β-carboxy group ofaspartic acid or the γ-carboxyl group of glutamic acid.

The peptide typically includes 3-12 (or more) amino acids. The selectionof particular amino acids will depend, at least in part, on the enzymeto be used for cleaving the peptide, as well as, the stability of thepeptide in vivo. One example of a suitable cleavable peptide isβ-AlaLeuAlaLeu. This can be combined with a stabilizing group to formsuccinyl-β-AlaLeuAlaLeu. Other examples of suitable cleavable peptidesare provided in the references cited above.

As one illustrative example, CD10, also known as neprilysin, neutralendopeptidase (NEP), and common acute lymphoblastic leukemia antigen(CALLA), is a type II cell-surface zinc-dependent metalloprotease.Cleavable substrates suitable for use with CD10 include LeuAlaLeu andIleAlaLeu. Other known substrates for CD10 include peptides of up to 50amino acids in length, although catalytic efficiency often declines asthe substrate gets larger.

Another illustrative example is based on matrix metalloproteases (MMP).Probably the best characterized proteolytic enzymes associated withtumors, there is a clear correlation of activation of MMPs within tumormicroenvironments. In particular, the soluble matrix enzymes MMP2(gelatinase A) and MMP9 (gelatinase B), have been intensively studied,and shown to be selectively activated during tissue remodeling includingtumor growth. Peptide sequences designed to be cleaved by MMP2 and MMP9have been designed and tested for conjugates of dextran and methotrexate(Chau et al., Bioconjugate Chem. 15:931-941 (2004)); PEG (polyethyleneglycol) and doxorubicin (Bae et al., Drugs Exp. Clin. Res. 29:15-23(2004)); and albumin and doxorubicin (Kratz et al., Bioorg. Med. Chem.Lett. 11:2001-2006 (2001)). Examples of suitable sequences for use withMMPs include, but are not limited to, ProValGlyLeuIleGly (SEQ. ID NO.102), GlyProLeuGlyVal (SEQ. ID NO. 103), GlyProLeuGlyIleAlaGlyGln (SEQ.ID NO. 104), ProLeuGlyLeu (SEQ. ID NO. 105), GlyProLeuGlyMetLeuSerGln(SEQ. ID NO. 106), and GlyProLeuGlyLeuTrpAlaGln (SEQ. ID NO. 107). (See,e.g., the previously cited references as well as Kline et al., Mol.Pharmaceut. 1:9-22 (2004) and Liu et al., Cancer Res. 60:6061-6067(2000).) Other cleavable substrates can also be used.

Yet another example is type II transmembrane serine proteases. Thisgroup of enzymes includes, for example, hepsin, testisin, and TMPRSS4.GlnAlaArg is one substrate sequence that is useful withmatriptase/MT-SP1 (which is over-expressed in breast and ovariancancers) and LeuSerArg is useful with hepsin (over-expressed in prostateand some other tumor types). (See, e.g., Lee et. al., J. Biol. Chem.275:36720-36725 and Kurachi and Yamamoto, Handbook of Proeolytic EnzymesVol. 2, 2^(nd) edition (Barrett A J, Rawlings N D & Woessner J F, eds)pp. 1699-1702 (2004).) Other cleavable substrates can also be used.

Another type of cleavable substrate arrangement includes preparing aseparate enzyme capable of cleaving the cleavable substrate that becomesassociated with the tumor or cells. For example, an enzyme can becoupled to a tumor-specific antibody (or other entity that ispreferentially attracted to the tumor or other target cell such as areceptor ligand) and then the enzyme-antibody conjugate can be providedto the patient. The enzyme-antibody conjugate is directed to, and bindsto, antigen associated with the tumor. Subsequently, the drug-cleavablesubstrate conjugate is provided to the patient as a prodrug. The drug isonly released in the vicinity of the tumor when the drug-cleavablesubstrate conjugate interacts with the enzyme that has become associatedwith the tumor so that the cleavable substrate is cleaved and the drugis freed. For example, U.S. Pat. Nos. 4,975,278; 5,587,161; 5,660,829;5,773,435; and 6,132,722, all of which are incorporated herein byreference, disclose such an arrangement. Examples of suitable enzymesand substrates include, but are not limited to, β-lactamase andcephalosporin derivatives, carboxypeptidase G2 and glutamic and asparticfolate derivatives.

In one embodiment, the enzyme-antibody conjugate includes an antibody,or antibody fragment, that is selected based on its specificity for anantigen expressed on a target cell, or at a target site, of interest. Adiscussion of antibodies is provided hereinabove. One example of asuitable cephalosporin-cleavable substrate is

Examples of Conjugates

The linkers and cleavable substrates of the invention can be used inconjugates containing a variety of partner molecules. Examples ofconjugates of the invention are described in further detail below.Unless otherwise indicated, substituents are defined as set forth abovein the sections regarding cytotoxins, linkers, and cleavable substrates.

A. Linker Conjugates

One example of a suitable conjugate is a compound of the formula:

wherein L¹ is a self-immolative linker; m is an integer 0, 1, 2, 3, 4,5, or 6; F is a linker comprising the structure:

wherein AA¹ is one or more members independently selected from the groupconsisting of natural amino acids and unnatural α-amino acids; c is aninteger from 1 to 20; L² is a self-immolative linker and comprises

wherein each R¹⁷, R¹⁸, and R¹⁹ is independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl and substituted or unsubstituted aryl, and w is an integerfrom 0 to 4; o is 1; L⁴ is a linker member; p is 0 or 1; X⁴ is a memberselected from the group consisting of protected reactive functionalgroups, unprotected reactive functional groups, detectable labels, andtargeting agents; and D comprises a structure:

wherein the ring system A is a member selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups; E and G aremembers independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a singlebond, or E and G are joined to form a ring system selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl; X is amember selected from O, S and NR²³; R²³ is a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, and acyl; R³ is OR¹¹, wherein R¹¹ is a member selected fromthe group consisting of H, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, monophosphates,diphosphates, triphosphates, sulfonates, acyl, C(O)R¹²R¹³, C(O)OR¹²,C(O)NR¹²R¹³, P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, R⁴,R^(4′), R⁵ and R^(5′) are members independently selected from the groupconsisting of H, substituted alkyl, unsubstituted alkyl, substitutedaryl, unsubstituted aryl, substituted heteroaryl, unsubstitutedheteroaryl, substituted heterocycloalkyl, unsubstitutedheterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶,OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, and O(CH₂)_(n)N(CH₃)₂, or anyadjacent pair of R⁴, R^(4′), R⁵ and R^(5′), together with the carbonatoms to which they are attached, are joined to form a substituted orunsubstituted cycloalkyl or heterocycloalkyl ring system having from 4to 6 members; wherein n is an integer from 1 to 20; R¹⁵ and R¹⁶ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, and substituted or unsubstitutedpeptidyl, wherein R¹⁵ and R¹⁶ together with the nitrogen atom to whichthey are attached are optionally joined to form a substituted orunsubstituted heterocycloalkyl ring system having from 4 to 6 members,optionally containing two or more heteroatoms; R⁶ is a single bond whichis either present or absent and when present R⁶ and R⁷ are joined toform a cyclopropyl ring; and R⁷ is CH₂—X¹ or —CH₂— joined in saidcyclopropyl ring with R⁶, wherein X¹ is a leaving group, wherein R¹¹links said drug to L¹, if present, or to F.

In some embodiments, the drug has structure (c) or (f) above. Onespecific example of a compound suitable for use as a conjugate is

Another example of a type of conjugate is a compound of the formula

wherein L¹ is a self-immolative linker; m is an integer 0, 1, 2, 3, 4,5, or 6; F is a linker comprising the structure:

wherein AA¹ is one or more members independently selected from the groupconsisting of natural amino acids and unnatural α-amino acids; c is aninteger from 1 to 20; L² is a self-immolative linker; o is 0 or 1; L⁴ isa linker member; p is 0 or 1; X⁴ is a member selected from the groupconsisting of protected reactive functional groups, unprotected reactivefunctional groups, detectable labels, and targeting agents; and Dcomprises a structure:

wherein the ring system A is a member selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups; E and G aremembers independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a singlebond, or E and G are joined to form a ring system selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl; X is amember selected from O, S and NR²³; R²³ is a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, and acyl; R³ is a member selected from the group consistingof (═O), SR¹¹, NHR¹¹ and OR¹¹, wherein R¹¹ is a member selected from thegroup consisting of H, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, monophosphates,diphosphates, triphosphates, sulfonates, acyl, C(O)R¹²R¹³, C(O)OR¹²,C(O)NR¹²R¹³, P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, in whichR¹², R¹³, and R¹⁴ are members independently selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen or carbon atom to which they are attached are optionally joinedto form a substituted or unsubstituted heterocycloalkyl ring systemhaving from 4 to 6 members, optionally containing two or moreheteroatoms; R⁴, R^(4′), R⁵ and R^(5′) are members independentlyselected from the group consisting of H, substituted alkyl,unsubstituted alkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵,OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, andO(CH₂)_(n)N(CH₃)₂, or any adjacent pair of R⁴, R^(4′), R⁵ and R^(5′),together with the carbon atoms to which they are attached, are joined toform a substituted or unsubstituted cycloalkyl or heterocycloalkyl ringsystem having from 4 to 6 members, wherein n is an integer from 1 to 20;R¹⁵ and R¹⁶ are independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, andsubstituted or unsubstituted peptidyl, wherein R¹⁵ and R¹⁶ together withthe nitrogen atom to which they are attached are optionally joined toform a substituted or unsubstituted heterocycloalkyl ring system havingfrom 4 to 6 members, optionally containing two or more heteroatoms;wherein at least one of R⁴, R^(4′), R⁵ and R^(5′) links said drug to L,if present, or to F, and comprises

wherein v is an integer from 1 to 6; and each R²⁷, R^(27′), R²⁸, andR28′ is independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted heterocycloalkyl; R⁶ is a single bond whichis either present or absent and when present R⁶ and R⁷ are joined toform a cyclopropyl ring; and R⁷ is CH₂—X¹ or —CH₂— joined in saidcyclopropyl ring with R⁶, wherein X¹ is a leaving group.

In some embodiment, the drug has structure (c) or (f) above. Onespecific example of a compound suitable for use as a conjugate is

where r is an integer in the range from 0 to 24.

Another example of a suitable conjugate is a compound of the formula

wherein L¹ is a self-immolative linker; m is an integer 0, 1, 2, 3, 4,5, or 6; F is a linker comprising the structure:

wherein AA¹ is one or more members independently selected from the groupconsisting of natural amino acids and unnatural α-amino acids; c is aninteger from 1 to 20; L³ is a spacer group comprising a primary orsecondary amine or a carboxyl functional group; wherein if L³ ispresent, m is 0 and either the amine of L³ forms an amide bond with apendant carboxyl functional group of D or the carboxyl of L³ forms anamide bond with a pendant amine functional group of D; o is 0 or 1; L⁴is a linker member, wherein L⁴ comprises

directly attached to the N-terminus of (AA¹)_(c), wherein R²⁰ is amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl, each R²⁵, R^(25′), R²⁶, andR^(26′) is independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted heterocycloalkyl; and s and t areindependently integers from 1 to 6; p is 1; X⁴ is a member selected fromthe group consisting of protected reactive functional groups,unprotected reactive functional groups, detectable labels, and targetingagents; and D comprises a structure:

wherein the ring system A is a member selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups; E and G aremembers independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a singlebond, or E and G are joined to form a ring system selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl; X is amember selected from O, S and NR²³; R²³ is a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, and acyl; R³ is a member selected from the group consistingof (═O), SR¹¹, NHR¹¹ and OR¹¹, wherein R¹¹ is a member selected from thegroup consisting of H, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, monophosphates,diphosphates, triphosphates, sulfonates, acyl, C(O)R¹²R¹³, C(O)OR¹²,C(O)NR¹²R¹³, P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, in whichR¹², R¹³, and R¹⁴ are members independently selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen or carbon atom to which they are attached are optionally joinedto form a substituted or unsubstituted heterocycloalkyl ring systemhaving from 4 to 6 members, optionally containing two or moreheteroatoms; R⁴, R^(4′), R⁵ and R^(5′) are members independentlyselected from the group consisting of H, substituted alkyl,unsubstituted alkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵,OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, andO(CH₂)_(n)N(CH₃)₂, or any adjacent pair of R⁴, R^(4′), R⁵ and R^(5′),together with the carbon atoms to which they are attached, are joined toform a substituted or unsubstituted cycloalkyl or heterocycloalkyl ringsystem having from 4 to 6 members, wherein n is an integer from 1 to 20;R¹⁵ and R¹⁶ are independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, andsubstituted or unsubstituted peptidyl, wherein R¹⁵ and R¹⁶ together withthe nitrogen atom to which they are attached are optionally joined toform a substituted or unsubstituted heterocycloalkyl ring system havingfrom 4 to 6 members, optionally containing two or more heteroatoms; R⁶is a single bond which is either present or absent and when present R⁶and R⁷ are joined to form a cyclopropyl ring; and R⁷ is CH₂—X¹ or —CH₂—joined in said cyclopropyl ring with R⁶, wherein X¹ is a leaving group,wherein at least one of R⁴, R^(4′), R⁵, R^(5′), R¹⁵ or R¹⁶ links saiddrug to L, if present, or to F.

In some embodiment, the drug has structure (c) or (f) above. Onespecific example of a compound suitable for use as conjugate is

where r is an integer in the range from 0 to 24.

Other examples of suitable compounds for use as conjugates include:

where R is

and r is an integer in the range from 0 to 24

Conjugates can also be formed using the drugs having structure (g), suchas the following compounds:

(where r is an integer in the range from 0 to 24.

Conjugates can also be formed using the drugs having the followingstructures:

Synthesis of such toxins, as well as details regarding their linkage toantibodies is disclosed in U.S. patent application having Ser. No.60/991,300.

B. Cleavable Linker Conjugates

One example of a suitable conjugate is a compound having the followingstructure:

wherein L¹ is a self-immolative spacer; m is an integer of 0, 1, 2, 3,4, 5, or 6; X² is a cleavable substrate; and D comprises a structure:

wherein the ring system A is a member selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups; E and G aremembers independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a singlebond, or E and G are joined to form a ring system selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl; X is amember selected from O, S and NR²³; R²³ is a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, and acyl; R³ is a member selected from the group consistingof (═O), SR¹¹, NHR¹¹ and OR¹¹, wherein R¹¹ is a member selected from thegroup consisting of H, substituted alkyl, unsubstituted alkyl,substituted heteroalkyl, unsubstituted heteroalkyl, monophosphates,diphosphates, triphosphates, sulfonates, acyl, C(O)R¹²R¹³, C(O)OR¹²,C(O)NR¹²R¹³, P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, in whichR¹², R¹³, and R¹⁴ are members independently selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen or carbon atom to which they are attached are optionally joinedto form a substituted or unsubstituted heterocycloalkyl ring systemhaving from 4 to 6 members, optionally containing two or moreheteroatoms; R⁶ is a single bond which is either present or absent andwhen present R⁶ and R⁷ are joined to form a cyclopropyl ring; and R⁷ isCH₂—X¹ or —CH₂— joined in said cyclopropyl ring with R⁶, wherein X¹ is aleaving group, R⁴, R^(4′), R⁵ and R^(5′) are members independentlyselected from the group consisting of H, substituted alkyl,unsubstituted alkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,unsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵,OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, OR¹⁵, CR¹⁵═NR¹⁶, andO(CH₂)_(n)N(CH₃)₂, or any adjacent pair of R⁴, R^(4′), R⁵ and R^(5′),together with the carbon atoms to which they are attached, are joined toform a substituted or unsubstituted cycloalkyl or heterocycloalkyl ringsystem having from 4 to 6 members, wherein n is an integer from 1 to 20;R¹⁵ and R¹⁶ are independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, andsubstituted or unsubstituted peptidyl, wherein R¹⁵ and R¹⁶ together withthe nitrogen atom to which they are attached are optionally joined toform a substituted or unsubstituted heterocycloalkyl ring system havingfrom 4 to 6 members, optionally containing two or more heteroatoms;wherein at least one of members R⁴, R^(4′), R⁵ and R^(5′) links saiddrug to L, if present, or to X², and is selected from the groupconsisting of

wherein R³⁰, R^(30′), R³¹, and R^(31′) are independently selected fromH, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and substituted or unsubstitutedheterocycloalkyl; and v is an integer from 1 to 6.

Examples of suitable cleavable linkers include β-AlaLeuAlaLeu and

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent disclosure, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of this disclosure. For example, a pharmaceuticalcomposition of this disclosure can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of this disclosure also can be administeredin combination therapy, i.e., combined with other agents. For example,the combination therapy can include an anti-CD22 antibody of the presentdisclosure combined with at least one other anti-cancer,anti-inflammatory or immunosuppressant agent. Examples of therapeuticagents that can be used in combination therapy are described in greaterdetail below in the section on uses of the antibodies of thisdisclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of this disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of this disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of this disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthis disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of this disclosure are dictated by anddirectly dependent on (a) the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-CD22antibody of this disclosure include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

For use in the prophylaxis and/or treatment of diseases related toabnormal cellular proliferation, a circulating concentration ofadministered compound of about 0.001 μM to 20 μM is preferred, withabout 0.01 μM to 5 μM being preferred.

Patient doses for oral administration of the compounds described herein,typically range from about 1 mg/day to about 10,000 mg/day, moretypically from about 10 mg/day to about 1,000 mg/day, and most typicallyfrom about 50 mg/day to about 500 mg/day. Stated in terms of patientbody weight, typical dosages range from about 0.01 to about 150mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and mosttypically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or3 mg/kg/day.

In at least some embodiments, patient doses that retard or inhibit tumorgrowth can be 1 μmol/kg/day or less. For example, the patient doses canbe 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1 μmol/kg/day or less(referring to moles of the drug). Preferably, the antibody-drugconjugate retards growth of the tumor when administered in the dailydosage amount over a period of at least five days. In at least someembodiments, the tumor is a human-type tumor in a SCID mouse. As anexample, the SCID mouse can be a CB17.SCID mouse (available fromTaconic, Germantown, N.Y.).

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-CD22 antibody of thisdisclosure preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of CD22⁺ tumors, a“therapeutically effective dosage” preferably inhibits cell growth ortumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to inhibit tumor growth can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit cell growth, suchinhibition can be measured in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of thisdisclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of this disclosure can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of this disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering mendicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of thisdisclosure can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of this disclosurecross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies, particularly the human antibodies, antibody compositionsand methods of the present disclosure have numerous in vitro and in vivodiagnostic and therapeutic utilities involving the diagnosis andtreatment of diseases and disorders involving CD22. For example, thesemolecules can be administered to cells in culture, in vitro or ex vivo,or to human subjects, e.g., in vivo, to treat, prevent and to diagnose avariety of disorders.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals include all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,cows, horses, chickens, amphibians, and reptiles. Preferred subjectsinclude human patients having disorders mediated by or modulated by CD22activity. When antibodies to CD22 are administered together with anotheragent, the two can be administered in either order or simultaneously.

Given the specific binding of the antibodies of this disclosure forCD22, the antibodies of this disclosure can be used to specificallydetect CD22 expression on the surface of cells and, moreover, can beused to purify CD22 via immunoaffinity purification.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of this disclosure in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-CD22 antibodies of this disclosurecan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/kg dose once everyfour weeks and adriamycin is intravenously administered as a 60-75 mg/mldose once every 21 days. Co-administration of human anti-CD22antibodies, or antigen binding fragments thereof, of the presentdisclosure with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells that would render them unreactive with the antibody.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be used as therapeutic agents.Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10⁸-10⁹ but will vary depending onthe therapeutic purpose. In general, the amount will be sufficient toobtain localization at the target cell, e.g., a tumor cell expressingCD22, and to effect cell killing by, e.g., phagocytosis. Routes ofadministration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) of this disclosureand/or effector cells armed with these compositions can be used inconjunction with chemotherapy. Additionally, combination immunotherapymay be used to direct two distinct cytotoxic effector populations towardtumor cell rejection. For example, anti-CD22 antibodies linked toanti-Fc-gamma RI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

Bispecific and multispecific molecules of this disclosure can also beused to modulate FcγR or FcγR levels on effector cells, such as bycapping and elimination of receptors on the cell surface. Mixtures ofanti-Fc receptors can also be used for this purpose.

The compositions (e.g., human, humanized, or chimeric antibodies,multispecific and bispecific molecules and immunoconjugates) of thisdisclosure which have complement binding sites, such as portions fromIgG1, -2, or -3 or IgM which bind complement, can also be used in thepresence of complement. In one embodiment, ex vivo treatment of apopulation of cells comprising target cells with a binding agent of thisdisclosure and appropriate effector cells can be supplemented by theaddition of complement or serum containing complement. Phagocytosis oftarget cells coated with a binding agent of this disclosure can beimproved by binding of complement proteins. In another embodiment targetcells coated with the compositions (e.g., human antibodies,multispecific and bispecific molecules) of this disclosure can also belysed by complement. In yet another embodiment, the compositions of thisdisclosure do not activate complement.

The compositions (e.g., human, humanized, or chimeric antibodies,multispecific and bispecific molecules and immunoconjugates) of thisdisclosure can also be administered together with complement.Accordingly, within the scope of this disclosure are compositionscomprising human antibodies, multispecific or bispecific molecules andserum or complement. These compositions are advantageous in that thecomplement is located in close proximity to the human antibodies,multispecific or bispecific molecules. Alternatively, the humanantibodies, multispecific or bispecific molecules of this disclosure andthe complement or serum can be administered separately.

The antibodies of this disclosure also can be used in combination withone or more additional therapeutic antibodies or other binding agents,such as Ig fusion proteins. Non-limiting examples of other antibodies orbinding agents with which an anti-CD22 antibody of this disclosure canbe administered in combination include antibodies or binding agents toCTLA-4, PSMA, CD30, IP-10, IFN-γ, CD70, PD-1, PD-L1, TNF, TNF-R, VEGF,VEGF-R, CCR5, IL-1, IL-18, IL-18R, CD19, Campath-1, EGFR, CD33, CD20,Her-2, CD25, gpIIb/IIIa, IgE, CD11a, α4 integrin.

Also within the scope of the present disclosure are kits comprisingantibody compositions of this disclosure (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain one ore moreadditional reagents, such as an immunosuppressive reagent, a cytotoxicagent or a radiotoxic agent, or one or more additional human antibodiesof this disclosure (e.g., a human antibody having a complementaryactivity which binds to an epitope in the CD22 antigen distinct from thefirst human antibody).

Accordingly, patients treated with antibody compositions of thisdisclosure can be additionally administered (prior to, simultaneouslywith, or following administration of a human antibody of thisdisclosure) with another therapeutic agent, such as a cytotoxic orradiotoxic agent, which enhances or augments the therapeutic effect ofthe human antibodies.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fcγ or Fcγ receptors by, for example, treating the subjectwith a cytokine. Preferred cytokines for administration during treatmentwith the multispecific molecule include of granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF).

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be used to target cellsexpressing CD22, for example for labeling such cells. For such use, thebinding agent can be linked to a molecule that can be detected. Thus,this disclosure provides methods for localizing ex vivo or in vitrocells expressing CD22. The detectable label can be, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

In a particular embodiment, this disclosure provides methods fordetecting the presence of CD22 antigen in a sample, or measuring theamount of CD22 antigen, comprising contacting the sample, and a controlsample, with a human monoclonal antibody, or an antigen binding portionthereof, which specifically binds to CD22, under conditions that allowfor formation of a complex between the antibody or portion thereof andCD22. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of CD22 antigen in the sample.

In yet another embodiment, immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxoins immunosuppressants, etc.) to cells which express CD22 bylinking such compounds to the antibody. For example, an anti-CD22antibody can be conjugated to any of the toxin compounds described inU.S. Pat. Nos. 6,281,354 and 6,548,530, U.S. patent publication Nos.20030050331, 20030064984, 20030073852, and 20040087497, or published inWO 03/022806. Thus, the invention also provides methods for localizingex vivo or in vivo cells expressing CD22 (e.g., with a detectable label,such as a radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor). Alternatively, the immunoconjugates can be used to killcells which have CD22 cell surface receptors by targeting cytotoxins orradiotoxins to CD22.

CD22 is known to be expressed on a large percentage of B cell lymphomasand also is known to be involved in regulating B cell activity such thatautoimmune disorders can be treated via targeting of CD22. Accordingly,the anti-CD22 antibodies (and immunoconjugates and bispecific molecules)of this disclosure can be used to modulate CD22 activity in each ofthese clinical situations.

Accordingly, in one aspect, the invention provides a method ofinhibiting growth of a CD22-expressing tumor cell. The method comprisescontacting the CD22-expressing tumor cell with the antibody, orantigen-binding portion thereof, of the invention such that growth ofthe CD22-expressing tumor cell is inhibited. Preferably, theCD22-expressing tumor cell is a B cell lymphoma, such as a non-Hodgkin'slymphoma. Other types of CD22-expressing tumor cells include Burkitt'slymphomas and B cell chronic lymphocytic leukemias.

In one embodiment of the method of inhibiting tumor cell growth, theantibody, or antigen-binding portion thereof, is conjugated to a partnermolecule, such as a therapeutic agent, such as a cytotoxin, radioisotopeor chemotherapeutic agent. In other embodiments, the antibody, orantigen-binding portion thereof, in administered in combination with oneor more additional anti-tumor agents. The antibody can be used incombination other cancer treatments, such as surgery and/or radiation,and/or with other anti-neoplastic agents, such as the anti-neoplasticagents discussed and set forth above, including chemotherapeutic drugsand other anti-tumor antigen antibodies, including but not limited to ananti-CD20 antibody (e.g., Rituxan®).

In another aspect, the invention provides a method of treating aninflammatory or autoimmune disorder in a subject. The method comprisesadministering to the subject the antibody, or antigen-binding portionthereof, of the invention such that the inflammatory or autoimmunedisorder in the subject is treated. Non-limiting examples of preferredautoimmune disorders include systemic lupus erythematosus and rheumatoidarthritis. Other examples of autoimmune disorders include inflammatorybowel disease (including ulcerative colitis and Crohn's disease), Type Idiabetes, multiple sclerosis, Sjogren's syndrome, autoimmune thyroiditis(including Grave's disease and Hashimoto's thyroiditis), psoriasis andglomerulonephritis. The antibody can be used alone or in combinationwith other anti-inflammatory or immunsuppresant agents, such asnon-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids (e.g.,prednisone, hydrocortisone), methotrexate, COX-2 inhibitors, TNFantagonists (e.g., etanercept, infliximab, adalimumab) andimmunosuppressants (such as 6-mercaptopurine, azathioprine andcyclosporine A).

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

Example 1 Generation of Human Monoclonal Antibodies Against CD22

Anti-CD22 human monoclonal antibodies were generated using transgenicmice that express human antibody genes, as follows.

Antigen

The antigens used to raise anti-CD22 antibodies were the extracellulardomain of human CD22 and the full-length CD22 protein expressed on CHOcells. To obtain the extracellular domain, a cDNA encoding human CD22(commercially available from Open Biosystems, Inc.) was used toconstruct an expression vector encoding the entire CD22β extracellulardomain (CD22 ECD) fused to a C-terminal hexahistidine tag. Aftertransfection of CHO cells and selection of stable transfectants bystandard techniques, CD22 ECD was purified from the cell culture mediumusing metal chelate chromatography. In addition, recombinant CHO cellswere created that expressed full-length CD22 on the cell surface bytransfecting the cells with an expression vector that contained thefull-length CD22 cDNA. After selection of the transfected cells, thosecells expressing high levels of CD22 on the cell surface were isolatedby fluorescent-activated cell sorting, based on reactivity with afluorescein-labeled anti-CD22 (commercially available fromBecton-Dickinson-Pharmingen).

Mouse Strains

Fully human monoclonal antibodies to CD22 were prepared using HCo7/HCo12and HCo12/Balbc strains of the transgenic HuMAb Mouse®, and the KM andKM-λHAC strains of transgenic transchromosomic mice, all of whichexpress human antibody genes.

In each of these mouse strains, the endogenous mouse kappa light chaingene has been homozygously disrupted as described in Chen et al. (1993)EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of PCT Publication WO01/09187. Each of these strains carries a human kappa light chaintransgene, KCo5 (as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851) and also contains the SC20 transchromosome,which carries the human Ig heavy chain locus, as described in PCTPublication WO 02/43478.

The HCo7 strain carries the HCo7 human heavy chain transgene asdescribed in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807. TheHCo12 strain carries the HCo12 human heavy chain transgene as describedin Example 2 of PCT Publication WO 01/09187.

The KM Mouse® strain is described in detail in U.S. Application No.20020199213.

The KM-λHAC strain is very similar to the KM strain in that theendogenous mouse heavy chain and kappa light chain loci have beendisrupted and the SC20 transchromosome and KCo5 transgene have beeinserted, but the KM-λHAC strain also carries a human artificialchromosome derived from human chromosome 22 that carries the humanlambda light chain locus. Thus, the KM-λHAC strain can express humanantibodies that utilize either a lambda light chain or a kappa lightchain. The KM-λHAC mice are also described in detail in U.S. ApplicationNo. 20060015958.

Immunization

To raise fully human monoclonal antibodies to CD22, animals of thestrains described above were immunized with recombinant human CD22 ECDand CD22-expressing CHO cells (prepared as described above for theantigen). General immunization schemes for the raising human antibodiesin mice strains carrying human antibody genes are described in, forexample, Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild,D. et al. (1996) Nature Biotechnology 14: 845-851 and PCT Publication WO98/24884. Mice were 10-12 weeks of age when the immunizations wereinitiated. Mice were immunized weekly intraperitoneally andsubcutaneously with 20 μg of CD22 ECD or 10⁷ transfected CHO cells withRIBI as adjuvant. The first two immunizations were performed with CD22ECD in RIBI adjuvant followed by six additional weekly immunizationsalternately using CD22 ECD or transfected cells (up to a total of 8immunizations). The immune response was monitored in blood harvested byretroorbital bleeds. The serum was screened by ELISA and FACS. Mice withadequate titer of anti-CD22 human IgG immunoglobulin were used forfusions. Mice were boosted once with CD22 ECD and once with CD22expressing CHO cells both intravenously and intraperitoneally on days −4and −3, respectively, before sacrifice and removal of the spleen.

Antibody Selection

To identify mice producing antibodies that bound CD22, sera fromimmunized mice were screened by flow cytometry for binding to CHO cellsexpressing human CD22 as well as to parental CHO cells. The sera werealso screened by flow cytometry (FACS) on human Daudi B cells, whichexpress CD22. Sera from all immunized mice were tested at a dilution of1:50 in the FACS experiment. After addition of diluted serum to thecells and incubation for 30 minutes at 37° C., cells were washed andbinding was detected with a PE-labeled anti-human IgG Ab. Flowcytometric analyses were performed using a FACSCalibur flow cytometry(Becton Dickinson, San Jose, Calif.). A murine anti-CD22 monoclonalantibody (M anti-CD22) was used as positive control in the experiment.All three mice tested exhibited titer to CHO-CD22 and CHO parental cells(CHO-S). Binding to CHO-S cells reflect the presence of antibodiesbinding to molecules other than CD22 on the surface of CHO cells. Thisresult was expected since mice were immunized with CHO transfectedcells. Titer to human Daudi cells was also detected in the three miceindicating the potential presence of antibodies specific to CD22 thatcould bind CD22 from a non-recombinant source.

Sera were further tested for binding to human CD22 ECD by ELISA.Briefly, microtiter plates were coated with purified CD22 ECD proteinproduced in CHO cells at 1-2.5 μg/ml in PBS (50 μl/well) for 2 hrs atroom temperature. The plate was then blocked with 300 μl/well of 1% BSAin PBS. Dilutions of sera (100 to 20000) from CD22-immunized mice wereadded to each well and incubated for 1-2 hours at ambient temperature.The plates were washed with PBS/Tween and incubated with agoat-anti-human IgG polyclonal antibody conjugated with horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates were developed with ABTS substrate (Sigma #A9941) in phosphatecitrate buffer with perborate (Sigma#P4922) or Moss ABTS-1000 andanalyzed by spectrophotometry at OD 415-495 nm. The three mice testedhad good titer of anti-CD22 antibodies and were therefore used forfusions.

Splenocyte Fusions

Mouse splenocytes were fused to a mouse myeloma cell line using electricfield based electrofusion using a Cyto Pulse large chamber cell fusionelectroporator (Cyto Pulse Sciences, Inc., Glen Burnie, Md.). Singlecell suspensions of splenocytes from immunized mice were fused toAg8.653 mouse myeloma cells (ATCC, CRL 1581) at a ratio of 1:1. Cellswere plated at approximately 2×10⁴/well in flat bottom microtiterplates. Plates were incubated for one week in DMEM high glucose mediumwith L-glutamine, sodium pyruvate (Mediatech, Inc., Herndon, Va.), 10%fetal Bovine Serum (Hyclone, Logan, Utah), 18% P388DI conditional media,5% Origen Hybridoma cloning factor (BioVeris, Gaithersburg, Va.), 4 mML-glutamine, 5 mM HEPES, 0.055 mM β-mercaptoethanol, 50 units/mlpenicillin, 50 mg/ml streptomycin and 1×Hypoxanthine-aminopterin-thymidine (HAT). After one week (day 7), HATgrowth media was replaced with medium containing HT. When extensivehybridoma growth occurred (day 10-11), hybridoma supernatants weretested for the presence of human IgG antibodies in an HTRF homogeneousassay. Fusions from KM-λHAC mice were screened for presence of human IgGbearing either a human kappa or a human lambda light chain. Positivehybridomas were then screened by FACS on Daudi cells and by ELISA forthe presence of CD22 specific human IgG antibodies. ELISA and FACSexperiments were performed as described above except that hybridomasupernatants (50-100 μl/well) were used instead of serum dilutions. Theantigen specific parental hybridoma lines were transferred to 24 wellplates, screened again and, if still positive for human IgG, subclonedonce by limiting dilution. The stable subclones were then scaled up invitro and antibodies were purified for further characterization.

Eighteen subclones were chosen for expansion for antibody purification.The isotypes of the expanded subclones included the following isotypes:IgG1; IgG4; IgG4/IgM; IgG1/IgM; IgG1/IgG2/λ; and IgG4/λ. Thirteen of thepurified antibodies were titrated by ELISA and FACS and each exhibitedspecific binding to human CD22 in both assays. Four subclones, 12C5,19A3, 16F7, 23C6, were selected for further structural analysis andsequencing.

Production of Recombinant Antibodies CD22.1 and CD22.2

The anti-CD22 antibody 19A3 was expressed in CHO cells as a human IgG1(f allotype) and the recombinant antibody was designated CD22.1. Inaddition, a variant of 19A3 designated CD22.2 was made in which themutation N57Q was made to remove the N-glycosylation site in the CDR2region of the V_(H) chain.

The V_(K) and V_(H) regions of 19A3 were amplified by PCR from cDNAclones and cloned into pCR4Blunt-TOPO (Invitrogen) to introducerestriction sites for cloning. Site directed mutagenesis was thenperformed to introduce an N57Q mutation into the heavy chain sequence toremove the N-glycosylation site in CDR2. The 19A3 V_(K) was subclonedinto the pICOFSneoK2.hCMV2.1 kb vector to produce vectorpICOFSneoK2.hCMV2.1 kb(CD22.19A3), and the V_(H) (both wild type andN57Q mutation) regions were subcloned into the pICOFSpurG vector toproduce vectors pICOFSpurG(CD22.19A3) and pICOFSpurG(CD22.19A3.VH.N57Q).These constructs for expression of light and heavy chain were linearizedand co-transfected into CHO-S cells using DMRIE-C (Invitrogen) andstable clones selected using standard techniques.

CHO-S clone 8G9 was chosen for CD22.1 expression. An overgrown cultureof this clone produced approximately 75 mg/liter of antibody. CHO-Sclone 17E11 was chosen for CD22.2 expression and yielded approximately413 mg/liter in overgrown culture. The structure and function of therecombinant antibodies CD22.1 and CD22.2 were then determined (seeExample 3 and Example 10, below).

Example 2 Structural Characterization of Human Anti-CD22 MonoclonalAntibodies

The cDNA sequences encoding the heavy and light chain variable regionsof the mAbs expressed by the 12C5, 19A3, 16F7, 23C6, CD22.1, CD22.2, 4G6and 21F6 clones described in Example 1 were sequenced using standard DNAsequencing techniques and the expressed proteins were characterized bystandard protein chemistry analysis.

Characterization of 12C5, 19A3, CD22.1, CD22.2, 16F7 and 23C6

The 12C5 clone was found to express an antibody comprising an IgG1 heavychain and a lambda light chain. The 19A3 clone was found to express anantibody comprising an IgG1 heavy chain and a kappa light chain. Theheavy and light chains of the recombinant mAb expressed by the 8G9 clonewere identical to those expressed by the 19A3 clone. The heavy chain ofthe recombinant mAb expressed by the 17E11 was identical to that of the19A3 with the exception of the introduced N57Q mutation. The light chainof the recombinant mAb expressed by the 17E11 clone was identical tothat expressed by the 19A3 clone. The 16F7 clone was found to expressantibodies comprising an IgG1 heavy chain and one of two different kappalight chains (referred to herein as V_(K).1 and V_(K).2, wherein 43% ofantibody protein comprised V_(K).1 and 57% of antibody protein comprisedV_(K).2). The 23C6 clone also was found to express antibodies comprisingan IgG1 heavy chain and one of two different kappa light chains (V_(K).1and V_(K).2, wherein 40% of antibody protein comprised V_(K).1 and 60%of antibody protein comprised V_(K).2). The 4G6 clone was found toexpress antibodies comprising an IgG1 heavy chain and one of twodifferent kappa light chains (referred to herein as V_(K).1 andV_(K).2). The 21F6 clone was found to express antibodies comprising oneof two different IgG1 heavy chains (referred to herein as V_(H)1 andV_(H)2) and a kappa light chain.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12C5 are shown in FIG. 1A and in SEQ ID NO:41 and 31,respectively.

The nucleotide and amino acid sequences of the lambda light chainvariable region of 12C5 are shown in FIG. 1B and in SEQ ID NO:45 and 35,respectively.

Comparison of the 12C5 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12C5 heavy chain utilizes a V_(H) segment from human germline V_(H)7-4.1, a D segment from the human germline 3-3, and a JH segment fromhuman germline JH 6B. Further analysis of the 12C5 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 1A and inSEQ ID NOs: 1, 5 and 9, respectively.

Comparison of the 12C5 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12C5 lambda light chain utilizes a V_(λ) segment from human germlineV_(λ) 2b2 and a Jλ segment from human germline JL 2. Further analysis ofthe 12C5 V_(λ) sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCDR3 regions as shown in FIG. 1B and in SEQ ID NOs: 13, 19 and 25,respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 19A3 are shown in FIG. 2A and in SEQ ID NOs:42 and 32,respectively. The nucleotide and amino acid sequences of the heavy chainvariable region of CD22.1 are identical to those of 19A3, and correspondto the nucleotide and amino acid sequences shown in FIG. 2A and SEQ IDNOs:42 and 32, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CD22.2 are shown in FIG. 2C and in SEQ ID NOs:61 and 60,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 19A3 are shown in FIG. 2B and in SEQ ID NO:46 and 36,respectively. The nucleotide and amino acid sequences of the light chainvariable regions of both CD22.1 and CD22.2 are identical to those of19A3, and correspond to the nucleotide and amino acid sequences shown inFIG. 2A and SEQ ID NOs:46 and 36, respectively.

Comparison of the 19A3/CD22.1 heavy chain immunoglobulin sequence to theknown human germline immunoglobulin heavy chain sequences demonstratedthat the 19A3 heavy chain utilizes a V_(H) segment from human germlineV_(H) 4-34, a D segment from the human germline 3-9, and a JH segmentfrom human germline JH 4B. Further analysis of the 19A3/CD22.1 V_(H)sequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown inFIG. 2A and in SEQ ID NOs: 2, 6 and 10, respectively

Comparison of the CD22.2 heavy chain immunoglobulin sequence to theknown human germline immunoglobulin heavy chain sequences demonstratedthat the CD22.2 heavy chain utilizes a V_(H) segment from human germlineV_(H) 4-34, a D segment from the human germline 3-9, and a JH segmentfrom human germline JH 4B. Further analysis of the CD22.2 V_(H) sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown inFIG. 2C and in SEQ ID NOs: 2, 60 and 10, respectively.

Comparison of the 19A3/CD22.1/CD22.2 light chain immunoglobulin sequenceto the known human germline immunoglobulin light chain sequencesdemonstrated that the 19A3/CD22.1/CD22.2 kappa light chain utilizes aV_(K) segment from human germline V_(K) L6 and a JK segment from humangermline JK 1. Further analysis of the 19A3/CD22.1/CD22.2 V_(K) sequenceusing the Kabat system of CDR region determination led to thedelineation of the light chain CDR1, CDR2 and CDR3 regions as shown inFIG. 2B and in SEQ ID NOs: 14, 20 and 26, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 16F7 are shown in FIG. 3A and in SEQ ID NO:43 and 33,respectively.

The nucleotide and amino acid sequences of the V_(K).1 kappa light chainvariable region of 16F7 are shown in FIG. 3B and in SEQ ID NO:47 and 37,respectively.

The nucleotide and amino acid sequences of the V_(K).2 kappa light chainvariable region of 16F7 are shown in FIG. 3C and in SEQ ID NO:48 and 38,respectively.

Comparison of the 16F7 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 16F7 heavy chain utilizes a V_(H) segment from human germline V_(H)5-51, a D segment from the human germline 3-10, and a JH segment fromhuman germline JH 3B. Further analysis of the 16F7 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 3A and inSEQ ID NOs: 3, 7 and 11, respectively.

Comparison of the 16F7 V_(K).1 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the 16F7 V_(K).1 kappa light chain utilizes a V_(K)segment from human germline V_(K) A27 and a JK segment from humangermline JK 1. Further analysis of the 16F7 V_(K) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CDR3 regions as shown in FIG. 3B and in SEQID NOs: 15, 21 and 27, respectively.

Comparison of the 16F7 V_(K).2 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the 16F7 V_(K).2 kappa light chain utilizes a V_(K)segment from human germline V_(K) A10 and a JK segment from humangermline JK 2. Further analysis of the 16F7 V_(K) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CDR3 regions as shown in FIG. 3C and in SEQID NOs: 16, 22 and 28, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 23C6 are shown in FIG. 4A and in SEQ ID NO:44 and 34,respectively.

The nucleotide and amino acid sequences of the V_(K).1 kappa light chainvariable region of 23C6 are shown in FIG. 4B and in SEQ ID NO:49 and 39,respectively.

The nucleotide and amino acid sequences of the V_(K).2 kappa light chainvariable region of 23C6 are shown in FIG. 4C and in SEQ ID NO:50 and 40,respectively.

Comparison of the 23C6 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 23C6 heavy chain utilizes a V_(H) segment from human germline V_(H)1-69, a D segment from the human germline 2-15, and a JH segment fromhuman germline JH 6B. Further analysis of the 23C6 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 4A and inSEQ ID NOs:4, 8 and 12, respectively.

Comparison of the 23C6 V_(K).1 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the V_(K).1 kappa light chain utilizes a V_(K) segmentfrom human germline V_(K) L6 and a JK segment from human germline JK 1.Further analysis of the 23C6 V_(K).1 sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CDR3 regions as shown in FIG. 4B and in SEQ ID NOs:17, 23 and29, respectively.

Comparison of the 23C6 V_(K).2 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the V_(K).2 kappa light chain utilizes a V_(K) segmentfrom human germline V_(K) L6 and a JK segment from human germline JK 1.Further analysis of the 23C6 V_(K).2 sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CDR3 regions as shown in FIG. 4B and in SEQ ID NOs:18, 24 and30, respectively.

FIG. 5A shows the alignment of the 12C5 heavy chain variable amino acidsequence (SEQ ID NO:31) with the germline V_(H) 7-4.1 encoded amino acidsequence (SEQ ID NO:51). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 5B shows the alignment of the 12C5 lambda light chain variableamino acid sequence (SEQ ID NO:35) with the germline V_(λ) 2b2 encodedamino acid sequence (SEQ ID NO:55). The CDR1, CDR2 and CDR3 regions aredelineated.

FIG. 6A shows the alignment of the 19A3 heavy chain variable amino acidsequence and the CD22.1 heavy chain variable amino acid sequence (SEQ IDNO:32) with the germline V_(H) 4-34 encoded amino acid sequence (SEQ IDNO:52). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 6B shows the alignment of the 19A3, CD22.1 and CD22.2 kappa lightchain variable amino acid sequences (all of which are identical to SEQID NO:36) with the germline V_(K) L6 encoded amino acid sequence (SEQ IDNO:56). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 6C shows the alignment of the CD22.2 heavy chain variable aminoacid sequence (SEQ ID NO:32) with the germline V_(H) 4-34 encoded aminoacid sequence (SEQ ID NO:52). The CDR1, CDR2 and regions are delineated.

FIG. 7A shows the alignment of the 16F7 heavy chain variable amino acidsequence (SEQ ID NO:33) with the germline V_(H) 5-51 encoded amino acidsequence (SEQ ID NO:53). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 7B shows the alignment of the 16F7 V_(K).1 kappa light chainvariable amino acid sequence (SEQ ID NO:37) with the germline V_(K) A27encoded amino acid sequence (SEQ ID NO:57). The CDR1, CDR2 and CDR3regions are delineated.

FIG. 7C shows the alignment of the 16F7 V_(K).2 kappa light chainvariable amino acid sequence (SEQ ID NO:38) with the germline V_(K) A10encoded amino acid sequence (SEQ ID NO:58). The CDR1, CDR2 and CDR3regions are delineated.

FIG. 8A shows the alignment of the 23C6 heavy chain variable amino acidsequence (SEQ ID NO:34) with the germline V_(H) 1-69 encoded amino acidsequence (SEQ ID NO:54). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 8B shows the alignment of the 23C6 V_(K).1 kappa light chainvariable amino acid sequence (SEQ ID NO:39) and the V_(K).2 kappa lightchain variable amino acid sequence (SEQ ID NO:40) with the germlineV_(K) L6 encoded amino acid sequence (SEQ ID NO:56). The CDR1, CDR2 andCDR3 regions are delineated.

Characterization of 4G6 and 21F6

The nucleotide and amino acid sequences of the heavy chain variableregion of 4G6 are shown in FIG. 17A and in SEQ ID NO:87 and 81,respectively.

The nucleotide and amino acid sequences of the V_(K).1 kappa light chainvariable region of 4G6 are shown in FIG. 17B and in SEQ ID NO:90 and 84,respectively.

The nucleotide and amino acid sequences of the V_(K).2 kappa light chainvariable region of 4G6 are shown in FIG. 17C and in SEQ ID NO:91 and 85,respectively.

Comparison of the 4G6 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 4G6 heavy chain utilizes a V_(H) segment from human germline V_(H)1-69, a D segment from the human germline 7-27, and a JH segment fromhuman germline JH 4B. Further analysis of the 4G6 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 17A and inSEQ ID NOs: 63, 66 and 69, respectively.

Comparison of the 4G6 V_(K).1 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the 16F7 V_(K).1 kappa light chain utilizes a V_(K)segment from human germline V_(K) L18 and a JK segment from humangermline JK 2. Further analysis of the 4G6 V_(K) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CDR3 regions as shown in FIG. 3B and in SEQID NOs: 72, 75 and 78, respectively.

Comparison of the 4G6 V_(K).2 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the 4G5 V_(K).2 kappa light chain utilizes a V_(K)segment from human germline V_(K) A27 and a JK segment from humangermline JK 4. Further analysis of the 4G6 V_(K) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CDR3 regions as shown in FIG. 17C and in SEQID NOs: 73, 76 and 79, respectively.

The nucleotide and amino acid sequences of the V_(H)0.1 heavy chainvariable region of 21F6 are shown in FIG. 18A and in SEQ ID NO:88 and82, respectively.

The nucleotide and amino acid sequences of the V_(H).2 heavy chainvariable region of 21F6 are shown in FIG. 18B and in SEQ ID NO:89 and83, respectively.

The nucleotide and amino acid sequences of the kappa light chainvariable region of 21F6 are shown in FIG. 18C and in SEQ ID NO:92 and86, respectively.

Comparison of the 21F6 V_(H).1 heavy chain immunoglobulin sequence tothe known human germline immunoglobulin heavy chain sequencesdemonstrated that the 21F6 heavy chain utilizes a V segment from humangermline V_(H) 4-34, a D segment from the human germline 3-9, and a JHsegment from human germline JH 4B. Further analysis of the 21F6 V_(H)sequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown inFIG. 3A and in SEQ ID NOs: 64, 67 and 70, respectively.

Comparison of the 21F6 V_(H).2 kappa light chain immunoglobulin sequenceto the known human germline immunoglobulin kappa light chain sequencesdemonstrated that the 21F6 V_(H).2 heavy chain utilizes a V_(H) segmentfrom human germline V_(H) 4-34, a D segment from the human germline 3-9,and a JH segment from human germline JH 4B. Further analysis of the 21F6V_(H) sequence using the Kabat system of CDR region determination led tothe delineation of the light chain CDR1, CDR2 and CDR3 regions as shownin FIG. 18B and in SEQ ID NOs: 65, 68 and 71, respectively.

Comparison of the 21F6 kappa light chain immunoglobulin sequence to theknown human germline immunoglobulin kappa light chain sequencesdemonstrated that the 21F6 kappa light chain utilizes a V_(K) segmentfrom human germline V_(K) L6 and a JK segment from human germline JK 4.Further analysis of the 21F6 V_(H) sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CDR3 regions as shown in FIG. 18C and in SEQ ID NOs: 74, 77 and80, respectively.

FIG. 19A shows the alignment of the 4G6 heavy chain variable amino acidsequence (SEQ ID NO:81) with the germline V_(H) 1-69 encoded amino acidsequence (SEQ ID NO:54). The CDR1, CDR2 and CDR3 regions are delineated.

FIG. 19B shows the alignment of the 4G6 V_(K).1 kappa light chainvariable amino acid sequence (SEQ ID NO:84) with the germline V_(K1) L18encoded amino acid sequence (SEQ ID NO:93). The CDR1, CDR2 and CDR3regions are delineated.

FIG. 18C shows the alignment of the 4G6 V_(K).2 kappa light chainvariable amino acid sequence (SEQ ID NO:85) with the germline V_(K) A27encoded amino acid sequence (SEQ ID NO:57). The CDR1, CDR2 and CDR3regions are delineated.

FIG. 20A shows the alignment of the 21F6 V_(H).1 heavy chain variableamino acid sequence (SEQ ID NO:82) with the germline V_(H) 4-34 encodedamino acid sequence (SEQ ID NO:52). The CDR1, CDR2 and CDR3 regions aredelineated.

FIG. 20B shows the alignment of the 21F6 V_(H).2 kappa light chainvariable amino acid sequence (SEQ ID NO:83) with the germline V_(H) 4-34encoded amino acid sequence (SEQ ID NO:52). The CDR1, CDR2 and CDR3regions are delineated.

FIG. 20C shows the alignment of the 21F6 kappa light chain variableamino acid sequence (SEQ ID NO:86) with the germline V_(K) L6 encodedamino acid sequence (SEQ ID NO:56). The CDR1, CDR2 and CDR3 regions aredelineated.

Recombinant Isotype Conversion

The 12C5, 19A3, 16F7, 23C6, CD22.1, CD22.2, 4G6 and 21F6 variableregions can be converted to full-length antibodies of any desiredisotype using standard recombinant DNA techniques. For example, DNAencoding the V_(H) and V_(L) regions can be cloned into an expressionvector that carries the heavy and light chain constant regions such thatthe variable regions are operatively linked to the constant regions.Alternatively, separate vectors can be used for expression of thefull-length heavy chain and the full-length light chain. Non-limitingexamples of expression vectors suitable for use in creating full-lengthantibodies include the pIE vectors described in U.S. Patent ApplicationNo. 20050153394 by Black.

Example 3 Binding Characteristics of Anti-CD22 Human MonoclonalAntibodies

In this example, binding affinities of the anti-CD22 antibodies 12C5,19A3, 16F7, 23C6 and 4G6 were examined by BIAcore analysis. Retention ofCD22 binding affinity by the 19A32 recombinant derivative antibodiesCD22.1 and CD22.2 was confirmed by means of ELISA analysis and FACS flowcytometry.

Epitope grouping of the 12C5, 19A3, 16F7, and 23C6 antibodies wasperformed by BIAcore analysis.

Finally, the CD22 domains to which the anti-CD22 antibodies of thepresent invention specifically bind were mapped using CHO cells thatexpressed a fusion protein containing only the amino terminal domains 1and 2 of CD22.

Binding Affinity and Kinetics

For determination of antibody affinity (K_(D)), experiments wereperformed in which the CD22 antigen was captured on a BIAcore chip usingan antibody to the His tag present on the antigen. Anti-His monoclonalantibody ab15149 (Abcam, Stock conc. 0.5 mg/mL) was coated on a CM5 chipat high density (3500 RUs), as recommended by the manufacturer. CD22 ECD(6.6 μg/mL) was captured on this surface for 60 sec at a flow-rate of 6μL/min. A single concentration (20 μg/mL) of anti-CD22 purified mAbs wasinjected over the captured antigen with an association time of 5 minutesand a dissociation time of 8 minutes, at a flow rate of 25 μg/mL. Thechip surface was regenerated after each cycle with 10 μL of 25 mM NaOH.Isotype controls were run on the chip and the data used to subtractnon-specific binding. All experiments were carried out on a BIAcore 3000surface plasmon resonance instrument, using BIAcore Control software v3.2. Data analysis was carried out using BiaEvaluation v. 3.2 software.

Fourteen of the selected anti-CD22 antibodies were tested in theaffinity experiment. The range of obtained affinity values for thetwelve antibodies was 0.07-9.95×10⁻⁹ M. The results for the fourantibodies structurally characterized in Example 2 are summarized belowin Table 1:

TABLE 1 BIAcore Binding Data for Anti-CD22 HuMAbs. Anti-CD22 BIAcoreAffinity (K_(D)) antibody 10⁻⁹ M Positive control 1.48 12C5 0.23 19A30.15 16F7 1.03 23C6 0.87 4G6 0.07

Retention of CD22 Binding Affinity by Recombinant Derivative AntibodiesCD22.1 and CD22.2

To determine whether CD22.1 and CD22.2 retained CD22 binding affinity,ELISA analysis was performed in which binding to CD22 ECD by CD22.1 andCD22.2 were compared to binding by the hybridoma-derived parentalantibody 19A3.

Recombinant CD22 extracellular domain (CD22 ECD) was coated on 96-wellELISA plates at 2 μg/ml, and after washing, blocking with 5% bovineserum albumin and washing again, the test antibodies were titrated from10 μg/ml downwards in 1:3 dilutions. After incubating for an hour,plates were washed, and goat anti-human IgG HRP conjugate was added toeach well. After a further one hour incubation plates were washed againand bound HRP conjugate detected through addition of TMB substrate,incubating until color developed and stopping with 1M hydrochloric acid.Absorbance was then read in a plate reader at 450 nm. Results (FIG. 12)clearly showed that the ability of CD22.1 and CD22.2 to bind to CD22 ECDwas equivalent to the parental antibody 19A3. This revealed thatexpression of the antibody in CHO cells was successful, and that themutation to remove the N-glycosylation site did not affect antigenbinding.

The ability of the 4G6 and 21F6 anti-CD22 monoclonal antibodies to bindCD22 ECD was also investigated, and found to bind specifically to CD22ECD.

FACS Analysis of CD22 Expressed on Cell Surfaces

The ability of CD22.1 and CD22.2 to bind CD22 was also confirmed by flowcytometry. Either CHO cells transfected with full-length CD22 (CHO-CD22)or Raji cells were resuspended in FACS buffer at 2×10⁵ cells/well, andafter pelleting the cells, antibody was titrated into the wells startingat 10 μg/ml and serially diluting 1:3. After mixing and incubating onice for 45 minutes, FACS buffer was added and the cells washed 4 times.After washing, goat anti-human IgG PE conjugate was added, and followinga further 30 minute incubation on ice, cells were again washed 4 timesbefore resuspending in FACS buffer and reading PE fluorescence on a FACSarray machine. Results (FIGS. 13 and 14) showed that CD22.1 and CD22.2bound strongly and equivalently to both the CHO-CD22 transfectants andRaji cells.

Binding of 4G6 and 21F6 to CD22 expressed on the surfaces of Raji cellsand CHO cells transformed with CD22 was also analyzed by FACS analysis.The results demonstrated that a high level of cell binding was obtained.See FIGS. 21, 22A and 22B. Neither antibody was able to bind to CHOcells absent transfection with CD22.

Epitope Grouping

Epitope binning was carried out by immobilizing selected antibodies onthe CM5 chip, based on standard immobilization protocols and flowingantibody-antigen complexes over the surface. Antibodies that hadoverlapping epitopes were competed out while those havingnon-overlapping epitopes gave rise to simultaneous binding to theantigen. An increasing signal denotes an epitope different from theantibody coated on the chip, and the opposite is true if signaldecreases. Antibodies that exhibited faster off-rate constants werechosen to be coated on the Biacore CM5 chip as they would facilitateeasier regeneration for repeated use of the chip. Purified anti-CD22antibodies were coated at high densities on different surfaces ofdifferent CM5 chips. Several rounds of iterative binning were carriedout until the distinct epitope groups were identified. Theconcentrations of antibodies varied between 50-200 μg/mL, which wereincubated with 4 nM-50 nM CD22 ECD for 2 hrs at RT. The incubatedcomplexes were passed over the antibody coated surfaces on each chip for2-6 min at 5-10 μL/min. Each cycle was regenerated by 15-30 mM NaOH. Thesignal obtained after 2-5 minutes of injection was plotted againstantibody concentration to determine the epitope groups. Antibodies weregrouped into various epitopes based on the above interpretation of theexperimental observation.

The results of the epitope grouping experiment were that four distinctepitope groups could be identified. Of fourteen anti-CD22 antibodiesexamined, five were found to be in Epitope Group 1, three were found tobe in Epitope Group 2, four were found to be in Epitope Group 3, one wasfound to be in Epitope Group 4 and one was found to be in Epitope Groups3 & 4, indicating that there is some overlap between Epitope Groups 3and 4. The results for the four antibodies structurally characterized inExample 2 are summarized below in Table 2:

TABLE 2 CD22 Epitope Groups Mapped by BIAcore Anti-CD22 antibody EpitopeGroup Positive control 1 12C5 4 19A3 1 16F7 3 23C6 2

Recognition of CD22 Amino Terminal Domains

The extracellular region of CD22 contains 7 immunoglobulin-type domains,of which the amino terminal 2 Ig-type domains may be particularlyimportant for CD22 ligand binding. In order to map which domains thehuman antibodies bound to, a recombinant construct was made in whichonly amino terminal domains 1 and 2 of the ECD were fused to the hingeand Fc regions of a mouse IgG heavy chain. The resultant fusion protein,designated CD22 d1d2-mFc, was expressed in CHO cells and purified foruse in binding assays. Human antibodies, previously shown to bind to theentire ECD of CD22, were then tested for their ability to bind to CD22d1d2-mFc.

Goat anti-mouse IgG was coated on 96-well ELISA plates at 5 μg/ml. Afterincubating overnight at 4° C., plates were washed and CD22 d1d2-mFc wasadded to each well at 2 μg/ml followed by incubation for 1 hour at roomtemperature. After plate washing, blocking with 5% bovine serum albuminand washing again, the test antibodies were added at 10 μg/ml. Afterincubating for an hour, plates were washed, and goat anti-human IgG HRPconjugate was added to each well. After a further one hour incubationplates were washed again and bound HRP conjugate detected throughaddition of TMB substrate, incubating until color developed and stoppingwith 1M hydrochloric acid. Absorbance was then read in a plate reader at450 nm.

Results (FIG. 15) showed that antibodies fell into two groups. Group 1represented by 23C6, 19A3, and the recombinant derivatives of 19A3,CD22.1 and CD22.2 bound to CD22 d1d2-mFc whereas group 2 represented by12C5 and 16F7 did not. This suggests that 12C5 and 16F7 recognizeepitopes on the CD22 ECD outside of the amino-terminal domains.

FACS Analysis of CD22 Expressed on Cell Surfaces

Binding of CD22.1 and CD22.2 to CD22 was also confirmed by flowcytometry. Either CHO cells transfected with full-length CD22 (CHO-CD22)or Raji cells were resuspended in FACS buffer at 2×10⁵ cells/well, andafter pelleting the cells, antibody was titrated into the wells startingat 10 μg/ml and serially diluting 1:3. After mixing and incubating onice for 45 minutes, FACS buffer was added and the cells washed 4 times.After washing, goat anti-human IgG PE conjugate was added, and followinga further 30 minute incubation on ice, cells were again washed 4 timesbefore resuspending in FACS buffer and reading PE fluorescence on a FACSarray machine. Results (FIGS. 13 and 14) showed that CD22.1 and CD22.2bound strongly and equivalently to both the CHO-CD22 transfectants andRaji cells.

Binding of 4G6 and 21F6 to CD22 expressed on the surfaces of Raji cellsand CHO cells transformed with CD22 was analyzed by FACS analysis. Theresults demonstrated that a high level of cell binding was obtained. SeeFIGS. 21, 22A and 22B. Neither antibody was able to bind to CHO cellsabsent transfection with CD22.

Example 4 Internalization of Anti-CD22 Antibodies

To determine the ability of the anti-CD22 human antibodies tointernalize into CD22-expressing cells, a Hum-ZAP internalization assaywas used with the Burkitt's lymphoma cell line Raji, which expressesCD22. The Hum-ZAP assay tests for internalization of a primary antibodythrough binding of a secondary antibody with affinity for human IgGconjugated to the toxin saporin.

The CD22-expressing Raji cells were seeded at 2.0×10⁴ cells/well (35μl/well). The anti-CD22 antibodies were added to the wells at 1.5 μg/ml(35 μl/well). Media alone was used as negative control. The Hum-ZAPreagent (Advanced Targeting Systems, San Diego, Calif., IT-22-25) wasthen added at a concentration of 3.0 μg/mL (35 μl/well) to half of thewells while the other half of the wells received media only. The plateswere incubated for 72 hours at 37° C. The cell viability was determinedusing CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison,Wis., #G7571). CellTiter-Glo buffer was mixed with CellTiter-Glosubstrate and 100 μL of the mixture was added to each well. Luminescencewas detected using Veritas Microplate Luminometer and Veritas software(Turner BioSystems, Sunnyvale, Calif.) per manufacturer directions.

Twelve different anti-CD22 antibodies were tested and all exhibited theability to internalize. The results for the four antibodies that werestructurally characterized in Example 2 are shown in the bar graph ofFIG. 9. As illustrated in FIG. 9, a marked decrease in Raji cellviability was observed in all wells containing anti-CD22 antibodies andHumZAP reagent, including wells with positive control, while cellviability was not affected in wells containing only the anti-CD22antibodies with no HumZAP reagent, demonstrating that the anti-CD22antibodies do not trigger cell killing on their own. As expected, inabsence of anti-CD22 antibodies, the negative control (referred to asmedia) did not show any cell killing in presence or absence of HumZAPreagent. These data demonstrate that the anti-CD22 antibodiesinternalize efficiently and release of saporin inside the cells isresponsible for the killing of CD22-expressing Raji cells in presence ofHumZAP reagent.

Example 5 Assessment of ADCC Activity of Anti-CD22 Antibodies

To determine the ability of the anti-CD22 human antibodies kill CD22+cell lines in the presence of effector cells via antibody dependentcellular cytotoxicity (ADCC), a fluorescence cytotoxicity assay wasused.

Human effector cells were prepared from whole blood as follows. Humanperipheral blood mononuclear cells were purified from heparinized wholeblood by standard Ficoll-paque separation. The cells were resuspended inRPMI1640 media containing 10% FBS (heat-inactivated) and 200 U/ml ofhuman IL-2 and incubated overnight at 37° C. The following day, thecells were collected and washed four times in culture media andresuspended at 1×10⁷ cells/ml. Target CD22+ cells were incubated withBATDA reagent (Perkin Elmer, Wellesley, Mass.) at 2.5 μl BATDA per 1×10⁶target cells/mL for 20 minutes at 37° C. The target cells were washedfour times, spun down and brought to a final volume of 1×10⁵ cells/ml.

The CD22+ cell lines Raji (human B lymphocyte Burkitt's lymphoma; ATCCAccession No. CCL-86) and Daudi (human B lymphocyte Burkitt's lymphoma;ATCC Accession No. CCL-213) were tested for antibody specific ADCC tothe human anti-CD22 monoclonal antibodies using the Delfia fluorescenceemission analysis as follows. Each target cell line (100 μl of labeledtarget cells, 10⁴ cells/well) was incubated with 50 μl of effector cells(10⁶ cells/well) and 50 μl of antibody (10 ug/ml final concentration). Atarget to effector ratio of 1:50 was used throughout the experiments. Inall studies, a human IgG1 isotype control was used as a negativecontrol. Cells were spun down at 2000 rpm and incubated for one hourincubation at 37° C. The supernatants were then collected, submitted tocentrifugation and 20 μl of supernatant was transferred to a flat bottomplate, to which 180 μl of Eu solution (Perkin Elmer, Wellesley, Mass.)was added and read in a RubyStar reader (BMG Labtech). The % lysis wascalculated as follows: (sample release−spontaneous release*100)/(maximumrelease−spontaneous release), where the spontaneous release is thefluorescence from wells which contain target cells plus effector cellsand maximum release is the fluorescence from wells containing targetcells and have been treated with 2% Triton-X.

For the Raji and Daudi cell ADCC assays, thirteen different anti-CD33antibodies were tested along with negative and positive controlantibodies (hIgG1 and CD20, respectively). In each assay, ten of thethirteen anti-CD22 antibodies exhibited levels of ADCC activity equal toor greater than the positive control antibody. The results for the fourantibodies that were structurally characterized in Example 2 are shownin the graphs of FIGS. 10A (Daudi cells) and 10B (Raji cells), whichshow % cell lysis. The data demonstrate that human anti-CD22 antibodiesthat exhibit ADCC activity can be selected, although the degree ofcytotoxicity of each antibody against CD22+ cells may differ dependingon which cell line is used as the target cell.

Example 6 Stimulation of Calcium Flux by Anti-CD22 Antibodies

To assess the ability of the anti-CD22 human antibodies to stimulatecalcium flux in CD22+ cells, the following calcium flux assay was used.Viable Ramos cells (ATCC Accession No. CRL-1596) were counted by trypanblue exclusion microscopy and diluted to 2×10⁶ cells/ml in RPMI+10% FBSculture media. From this cell suspension, 2×10⁵ cells (100 μl/well) wasdispensed into to all the wells of a Poly D-Lysine surface black withclear bottom 96 well plate (Corning #3667). Loading dye (MolecularDevices catalog # R7181) was added to the cell suspension, 100 μl/well.The plate was centrifuges at 1100 rpm for 4 minutes and then incubatedat 37° C. for 30 minutes. Five-fold dilution series of anti-CD22antibodies and human IgG1 isotype control, from 50 μg/ml to 40 ng/ml,were prepared in Component B (Molecular Devices # R7181)+0.1% BSAbuffer. Antibodies were dispensed in triplicate into rows A-F of thepreviously prepared 96-well plate. Component B+0.1% BSA was dispensedinto all the wells of rows G&H on the assay plate. Calcium flux wasassayed using a Flex Station (Molecular Devices), adding 22 μl reagentper well to the assay plate at 17 seconds. Data was analyzed as FIMax-Min and plotted vs. antibody concentration using GraphPad™ PRISM,non-linear regression, sigmoidal dose response, variable slope.

Seventeen different anti-CD22 human antibodies were evaluated in theassay. The results showed that none of the seventeen anti-CD22antibodies tested stimulated significant calcium flux, as compared to ahuman IgG1 isotype control or buffer alone. Ramos cells were previouslydemonstrated to flux calcium in response to BCR stimulation with goatanti-human IgM F(ab′)₂.

Example 7 Modulation of BCR Stimulation-Induced Effects by Anti-CD22Antibodies

In this example, the ability of immobilized anti-CD22 antibody tomodulate B Cell Receptor (BCR) stimulation-induced effects was examined.In the assay, anti-CD22 human antibodies and human IgG1 isotype controlwere diluted to 5 μg/ml in RPMI+10% FBS and dispensed 100 μl/well intriplicate into Microlite 1 Flat Bottom plates (Corning #7416).Following overnight incubation at 4° C., the plates were washed oncewith cold PBS, then once with RPMI 1640 (Mediatech)+10% FBS (GIBCO).Viable Ramos cells (ATCC Accession No. CRL-1596) were counted by trypanblue exclusion microscopy and diluted to 2×10⁵ cells/ml in RPMI+10% FBS.20,000 cells (50 μl/well) were dispensed into the antibody coated96-well plates. Anti-human IgM F(ab′)₂ (Jackson catalog #109-006-129)was diluted to 5 μg/ml in RPMI+10% FBS and 100 μl/well was dispensed fora final concentration of 2.5 μg/ml. The assay plates were incubated 72hours. Cell viability was assayed with the addition of CellTiter-Gloreagent (Promega G7571), 100 μl/well, for 10 minutes. Luminescence wasmeasured using Luminescence Test 1 plate Nunc96 on Pherastar GMBLabtech. Data was analyzed as % cell death relative to the human IgG1isotype control, which represented 100% cell viability.

Seventeen different anti-CD22 antibodies were tested in the assay andthis panel exhibited a broad range of activity, ranging from an observed% cell death value of approximately 80% to an observed % cell deathvalue approximately equal to the isotype control, depending on theparticular antibody. The results for the four antibodies that werestructurally characterized in Example 2 are shown in the bar graph ofFIG. 11, which shows that immobilized 19A3, 23C6 and 12C5 each potentlyenhanced the anti-proliferative effects of BCR stimulation cell deathwhereas 16F7 did not differ significantly from the isotype control

Example 8 Effects of Anti-CD22 Antibodies on Cell Proliferation

In this example, the direct effects of soluble anti-CD22 antibodies oncell proliferation, with or without antibody cross-linking, wasexamined. In the assay used, viable Ramos cells (ATCC Accession No.CRL-1596) were counted by trypan blue exclusion microscopy and dilutedto 2×10⁵ cells/ml in RPMI+10% FBS. 20,000 cells (50 μl/well) weredispensed into 96-well culture treated plates. Cross-linking antibodygoat anti-human IgG Fc (Rockland catalog #709-1117) was diluted to 80μg/ml in RPMI+10% FBS and 25 μl/well was dispensed into half of theRamos cell assay plate. Diluent alone was dispensed (25 μl/well) intothe remainder of the plate. Anti-CD22 human antibodies and human IgG1isotype control were diluted to 20 μg/ml in RPMI+10% FBS and 25 μl/wellwas dispensed in triplicate to both Ramos plus cross-linker and Ramosplus diluent containing wells such that the final antibodyconcentrations were 5 μg/ml anti-CD22+/−20 μg/ml cross-linking antibody.Assay plates were incubated at 37° C. for 72 hours. Cell viability wasassayed with the addition of CellTiter-Glo reagent (Promega G7571), 100μl/well, for 10 minutes. Luminescence was measured using LuminescenceTest 1 plate Nunc96 on Pherastar GMB Labtech. Data was analyzed as %growth inhibition relative to the human IgG1 isotype control, whichrepresented 0% inhibition.

Seventeen different anti-CD22 human antibodies were evaluated in theassay. The results showed that none of the seventeen anti-CD22antibodies tested significantly altered the rate of Ramos cellproliferation, either when the antibodies were not cross-linked or whenthey were cross-linked. These results indicate that none of theanti-CD22 antibodies have a direct anti-proliferative effect on Ramoscells, even if the antibody is cross-linked.

Example 9 Assessment of CDC Activity of Anti-CD22 Antibodies

In this example, the ability of soluble anti-CD22 human antibodies tomediate complement dependent cytotoxicity (CDC) was examined. In theassay used, viable Ramos cells (ATCC Accession No. CRL-1596) werecounted by trypan blue exclusion microscopy and diluted to 1×10⁶cells/ml in CDC buffer (RPMI 1640+0.1% BSA+20 mM HEPES+1% Pen/strep).The cell suspension was dispensed as 50,000 cells (50 μl/well) in a96-well flat-bottomed tissue culture treated plate. Human complement(Quidel catalog # A113) was heat-inactivated by incubating 1 hr at 56°C. Active and heat-inactivated complement were each diluted 1:3 in CDCbuffer and were dispensed 50 μl/well into the Ramos assay plates.Anti-CD22 human antibodies, human IgG isotype control and an anti-CD20positive control antibody were each diluted to 40 μg/ml in CDC buffer.Diluted antibodies were dispensed 50 μl/well in duplicate into the Ramosassay plates with both active and heat-inactivated complement such thatthe final concentration of antibody was 10 μg/ml. The assay plates wereincubated 2 hrs at 37° C. To analyze cell viability, alamar blue reagent(BioSource catalog # DAL1100) was added 50 μl/well and the plates wereincubated a further 21 hrs at 37° C. Cell viability was assayed as beingproportional to fluorescence measure using the SPECTROMAX GEMINIfluorescence plate reader (Molecular Devices S/N G 02243).

Eighteen different anti-CD22 antibodies were tested, along with, as apositive control, an anti-CD20 antibody known to exhibit robustcytotoxicity in the presence of active but not heat inactivatedcomplement. The results showed that none of the anti-CD22 antibodiestested exhibited significant CDC activity as compared to the human IgGisotype control.

Example 10 Inhibition of Solid Tumor Cell Proliferation In Vivo byAnti-CD22 Antibody-Drug Conjugates

To determine whether drug conjugates of CD22.1 and CD22.2 could be madewhich could effectively inhibit proliferation of an established solidtumor in vivo, the anti-CD22 recombinant antibodies CD22.1 and CD22.2were conjugated to the cytotoxic drug Cytotoxin A and the efficacy ofthe resulting ADC compounds were examined using a Ramos subcutaneoustumor cell model.

Conjugation of CD22.1 and CD22.2 to Cytotoxic Compound Cytotoxin A

CD22.1 and CD22.2 were concentrated to approximately 5 mg/ml, bufferexchanged into 20 mM phosphate buffer, 50 mM NaCl, 2 mM DTPA, 3%Glycerol, pH 7.5 and thiolated with a 14-fold molar excess of2-Imminothiolane for 60 minutes at room temperature. Followingthiolation, the antibody was buffer exchanged into 50 mM HEPES buffer,containing 5 mM glycine, 2 mM DTPA, and 0.5% Povidone (10 K) pH 5.5.Thiolation was quantified with 4, 4″-dithiodipyridine by measuringthiopyridine release at 324 nM. Conjugation was achieved by addition ofCytotoxin A at a 3:1 molar ratio of Cytotoxin A to thiols. Incubationwas at room temperature for 60 minutes followed by blocking of anyresidual thiols by the addition of a 10:1 molar ratio ofN-ethylmaleimide to thiols to the reaction mix.

The resulting conjugates were purified by ion-exchange chromatography.Each reaction mix was filtered and loaded onto an SP-Sepharose HighPerformance column equilibrated with Buffer A (50 mM HEPES, 5 mMGlycine, 0.5% Povidone (10K), pH 5.5). Antibody conjugates were elutedwith 24% Buffer B (50 mM HEPES, 5 mM Glycine, 1M NaCl, 0.5% Povidone(10K), pH 5.5). Fractions containing monomeric antibody-Cytotoxin Aconjugate were pooled and dialyzed against 50 mM HEPES, 5 mM glycine,100 mM NaCl, 0.5% Povidone (10K), pH 6.0. Substitution ratios weredetermined by measuring absorbance at 280 and 340 nm, and the conjugatesanalyzed by SEC-HPLC.

CD22.1-Cytotoxin A conjugate was made with a substitution ratio of 1.7,and CD22.2 conjugate was made with a substitution ratio of 1.6.

In Vivo Efficacy of Anti-CD22 Antibody-Drug Conjugates CD22.1-CytotoxinA and CD22.2-Cytotoxin A

SCID mice were implanted subcutaneously with Raji cells at 10 millioncells per mouse in matrigel, and tumors allowed to grow until wellestablished with a median size of approx. 190 mm³. Groups of 8 mice werethen treated with a single dose of either CD22.1-Cytotoxin A antibodyconjugate, CD22.2-Cytotoxin A conjugate, a control human IgG-Cytotoxin Aconjugate which did not bind to Raji cells, or with vehicle alone. Tumorsize was monitored for 63 days post dosing, or until animals wereeuthanized due to tumor growth beyond 1500 mm³. CD22.1-Cytotoxin A wasadministered at 0.18 μmol/kg drug equivalent, and CD22.2-Cytotoxin A andcontrol ab-Cytotoxin A were administered at 0.3 μmol/kg drug equivalent.Results demonstrated good anti-tumor efficacy for both CD22.1 and CD22.2conjugates (FIG. 16).

SUMMARY OF SEQUENCE LISTING

SEQ ID NO: SEQUENCE SEQ ID NO: SEQUENCE 1 V_(H) CDR1 a.a. 12C5 31 V_(H)a.a. 12C5 2 V_(H) CDR1 a.a. 19A3 32 V_(H) a.a. 19A3 3 V_(H) CDR1 a.a.16F7 33 V_(H) a.a. 16F7 4 V_(H) CDR1 a.a. 23C6 34 V_(H) a.a. 23C6 5V_(H) CDR2 a.a. 12C5 35 V_(K) a.a. 12C5 6 V_(H) CDR2 a.a. 19A3 36 V_(K)a.a. 19A3 7 V_(H) CDR2 a.a. 16F7 37 V_(K).1 a.a. 16F7 8 V_(H) CDR2 a.a.23C6 38 V_(K).2 a.a. 16F7 39 V_(K).1 a.a. 23C6 9 V_(H) CDR3 a.a. 12C5 40V_(K).2 a.a. 23C6 10 V_(H) CDR3 a.a. 19A3 11 V_(H) CDR3 a.a. 16F7 41V_(H) n.t. 12C5 12 V_(H) CDR3 a.a. 23C6 42 V_(H) n.t. 19A3 43 V_(H) n.t.16F7 13 V_(K) CDR1 a.a. 12C5 44 V_(H) n.t. 23C6 14 V_(K) CDR1 a.a. 19A315 V_(K).1 CDR1 a.a. 16F7 45 V_(K) n.t. 12C5 16 V_(K).2 CDR1 a.a. 16F746 V_(K) n.t. 19A3 17 V_(K).1 CDR1 a.a. 23C6 47 V_(K).1 n.t. 16F7 18V_(K).2 CDR1 a.a. 23C6 48 V_(K).2 n.t. 16F7 49 V_(K).1 n.t. 23C6 19V_(K) CDR2 a.a. 12C5 50 V_(K).2 n.t. 23C6 20 V_(K) CDR2 a.a. 19A3 21V_(K).1 CDR2 a.a. 16F7 51 V_(H) 7-4.1 germline a.a. 22 V_(K).2 CDR2 a.a.16F7 52 V_(H) 4-34 germline a.a. 23 V_(K).1 CDR2 a.a. 23C6 53 V_(H) 5-51germline a.a. 24 V_(K).2 CDR2 a.a. 23C6 54 V_(H) 1-69 germline a.a. 25V_(K) CDR3 a.a. 12C5 55 V_(λ) 2b2 germline a.a. 26 V_(K) CDR3 a.a. 19A356 V_(K) L6 germline a.a. 27 V_(K).1 CDR3 a.a. 16F7 57 V_(K) A27germline a.a. 28 V_(K).2 CDR3 a.a. 16F7 58 V_(K) A10 germline a.a. 29V_(K).1 CDR3 a.a. 23C6 30 V_(K).2 CDR3 a.a. 23C6 59 human CD22(NP_001762) 60 V_(H) CDR2 a.a. CD22.2 61 V_(H) a.a. CD22.2 62 V_(H) n.t.CD22.2 63 V_(H) CDR1 a.a. 4G6 81 V_(H) a.a. 4G6 64 V_(H1) CDR1 a.a.21F682 V_(H1) a.a. 21F6 65 V_(H2) CDR1 a.a. 21F6 83 V_(H2) a.a. 21F6 66V_(H) CDR2 a.a. 4G6 84 V_(K1) a.a. 4G6 67 V_(H1)CDR2 a.a. 21F6 85 V_(K2)a.a. 4G6 68 V_(H2) CDR2 a.a. 21F6 86 V_(K) a.a. 21F6 69 V_(H) CDR3 a.a.4G6 70 V_(H1) CDR3 a.a. 21F6 87 V_(H) n.t. 4G6 71 V_(H2) CDR3 a.a. 21F688 V_(H1) n.t. 21F6 89 V_(H2) n.t. 21F6 72 V_(K1) CDR1 a.a. 4G6 73V_(K2) CDR1 a.a. 4G6 90 V_(K1) n.t. 4G6 74 V_(K) CDR1 a.a. 21F6 91V_(K2) n.t. 4F6 92 V_(K2) n.t. 21F6 75 V_(K1) CDR2 a.a. 4G6 76 V_(K2)CDR2 a.a. 4G6 93 V_(K1) L18 germline a.a. 77 V_(K) CDR2 a.a. 21F6 94Peptide Linker 78 V_(K1) CDR3 a.a. 4G6 95 Peptide Linker 79 V_(K2) CDR3a.a. 4G6 96 Peptide Linker 80 V_(K) CDR3 a.a. 21F6 97 Peptide Linker 98Peptide Linker 99 Peptide Linker 100 Peptide Linker 101 Peptide Linker102 Peptide Linker 103 Peptide Linker 104 Peptide Linker 105 PeptideLinker 106 Peptide Linker 107 Peptide Linker 108 12C5 JH6b germline 11821F6 4-34 germline VH1 109 JL2 germline 119 21F6 JH4b germline VH1 110JK1 germline 120 21F6 4-34 germline VH2 111 JK4b germline 121 21F6 JH4bgermline VH2 112 JK3b germline 122 21F6 VK L6 germline 113 JK1 germline123 21F6 VK JK4 germline 114 JK2 germline 124 4G6 VH 1-69 germline 1152-15 germline 125 4G6 VH JH4b germline 116 JK1 germline 126 4G6 VK1 JK2germline 117 JH4b germline 127 4G6 VK2 A27 germline 128 4G6 VK2 JK4germline

1. A method of treating a CD22-expressing cancer, comprisingadministering to a subject a human monoclonal antibody whichspecifically binds to CD22, or antigen-binding portion thereof, in anamount effective to treat the cancer.
 2. The method of claim 1, whereinthe cancer is a B cell lymphoma.
 3. The method of claim 2, wherein the Bcell lymphoma is a non-Hodgkin's lymphoma.
 4. The method of claim 1,wherein the cancer is selected from Burkitt's lymphoma and B cellchronic lymphocytic leukemia.
 5. The method of claim 1, wherein thesubject is human.
 6. A method of inhibiting growth of a CD22-expressingtumor cell, the method comprising contacting the CD22-expressing tumorcell with a human monoclonal antibody which specifically binds to CD22,or antigen-binding portion thereof, such that growth of theCD22-expressing tumor cell is inhibited. 7-8. (canceled)
 9. The methodof claim 1, wherein the antibody, or antigen-binding portion thereof,comprises: (a) a heavy chain variable region CDR1 comprising SEQ IDNO:2; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:6 orSEQ ID NO:60; (c) a heavy chain variable region CDR3 comprising SEQ IDNO: 10; (d) a light chain variable region CDR1 comprising SEQ ID NO:14;(e) a light chain variable region CDR2 comprising SEQ ID NO:20; and (f)a light chain variable region CDR3 comprising SEQ ID NO:26.
 10. Themethod of claim 9, wherein the antibody, or antigen-binding portionthereof, comprises: (a) a heavy chain variable region CDR1 comprisingSEQ ID NO:2; (b) a heavy chain variable region CDR2 comprising SEQ IDNO:6; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;(d) a light chain variable region CDR1 comprising SEQ ID NO:14; (e) alight chain variable region CDR2 comprising SEQ ID NO:20; and (f) alight chain variable region CDR3 comprising SEQ ID NO:26.
 11. The methodof claim 9, wherein the antibody, or antigen-binding portion thereof,comprises: (a) a heavy chain variable region CDR1 comprising SEQ IDNO:2; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:60;(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10; (d) alight chain variable region CDR1 comprising SEQ ID NO:14; (e) a lightchain variable region CDR2 comprising SEQ ID NO:20; and (f) a lightchain variable region CDR3 comprising SEQ ID NO:26.
 12. The method ofclaim 9, wherein the antibody, or antigen-binding portion thereof,comprises: (a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:32 or 61; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:36.
 13. The method ofclaim 12, wherein the antibody, or antigen-binding portion thereof,comprises: (a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:32; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:36.
 14. The method ofclaim 12, wherein the antibody, or antigen-binding portion thereof,comprises: (a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:61; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:36.
 15. The method ofclaim 1, wherein the antibody, or antigen-binding portion thereof,cross-competes for binding to CD22 with a reference antibody, whereinthe reference antibody comprises: (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:32 or 61; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO:36.
 16. The method of claim 1, wherein the antibody is a full-lengthantibody of an IgG1 isotype or an IgG4 isotype.
 17. The method of claim1, wherein the antibody or antigen-binding portion thereof, is linked toa therapeutic agent.
 18. The method of claim 17, wherein the therapeuticagent is a cytotoxin or a radioactive agent.
 19. The method of claim 17,wherein the therapeutic agent is conjugated to the antibody by achemical linker.
 20. The method of claim 19, wherein the chemical linkeris selected from the group consisting of peptidyl linkers, hydrazinelinkers, and disulfide linkers.
 21. The method of claim 1, wherein theantibody is administered by a route selected from subcutaneously,intravenously, intramuscularly, intradermally, and intraperitoneally.22. The method of claim 1, further comprising administering to thesubject a second anti-neoplastic agent.